Matrix composed of a naturally-occurring protein backbone cross linked by a synthetic polymer and methods of generating and using same

ABSTRACT

A method of treating a disorder characterized by tissue damage is provided. The method comprising providing to a subject in need-thereof a composition which comprises a synthetic polymer attached to denatured fibrinogen or a therapeutic portion of the fibrinogen, the composition being formulated for releasing the therapeutic portion of the fibrinogen in a pharmacokinetically regulated manner, thereby treating the disorder characterized by tissue damage or malformation.

RELATED APPLICATIONS

This is a continuation-in-part of PCT Patent Application No.PCT/IL2004/001136 filed Dec. 15, 2004, which claims the benefit of U.S.Provisional Patent Application No. 60/530,917 filed Dec. 22, 2003. Thecontents of the above applications are all incorporated by reference.

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to compositions and methods for treatingdisorders associated with tissue damage, loss, or malformation.

Tissue engineering, i.e., the generation of new living tissues in vitro,is widely used to replace diseased, traumatized or other unhealthytissues. The classic tissue engineering approach utilizes living cellsand a basic scaffold for cell culture (Langer and Vacanti, 1993; Neremand Seliktar, 2001). Thus, the scaffold structure attempts to mimic thenatural structure of the tissue it is replacing and to provide atemporary functional support for the cells (Griffith L G, 2002).

Tissue engineering scaffolds are fabricated from either biologicalmaterials or synthetic polymers. Synthetic polymers such as polyethyleneglycol (PEG), Hydroxyapatite/polycaprolactone (HA/PLC), polyglycolicacid (PGA), Poly-L-lactic acid (PLLA), Polymethyl methacrylate (PMMA),polyhydroxyalkanoate (PHA), poly-4-hydroxybutyrate (P4HB), polypropylenefumarate (PPF), polyethylene glycol-dimethacrylate (PEG-DMA),beta-tricalcium phosphate (beta-TCP) and nonbiodegradablepolytetrafluoroethylene (PTFE) provide precise control over the physicalproperties of the material (Drury and Mooney, 2003).

Common scaffold fabrication methods are based on foams of syntheticpolymers. However, cell migration into the depth of synthetic scaffoldsis limited by the lack of oxygen and nutrient supply. To overcome suchlimitations, new approaches utilizing solid freeform fabrications andinternal vascular architecture have been developed (Reviewed in SachlosE and Czemuszka J T, 2003; Eur. Cell Mater. 5: 29-39). Likewise,freeze-drying methods are also employed to create uniquethree-dimensional architectures with distinct porosity and permeability.However, creating pores into these materials is an aggressive procedure,often involving the use of toxic conditions which eliminate thepossibility of pre-casting tissue constructs with living cells.Therefore, many of the prefabricated materials are subject to unevencell seeding and inhomogeneous populations of cells within theconstructs. Furthermore, the materials are generally degraded unevenlyduring the tissue cultivation process, creating a highly anisotropictissue with altered growth kinetics.

Scaffolds made of PEG are highly biocompatible (Merrill and Salzman,1983) and exhibit versatile physical characteristics based on theirweight percent, molecular chain length, and cross-linking density(Temenoff J S et al., 2002). In addition, PEG hydrogels are capable of acontrolled liquid-to-solid transition (gelation) in the presence of cellsuspension (Elbert and Hubbell, 2001). Moreover, the PEG gelation (i.e.,PEGylation) reaction can be carried out under non-toxic conditions inthe presence of a photoinitiator (Elisseeff J et al., 2000; Nguyen andWest, 2002) or by mixing a two-part reactive solution of functionalizedPEG and cross-linking constituents (Lutolf and Hubbell, 2003).

However, while the abovementioned synthetic polymers enable precisecontrol over the scaffold material, they often provide inadequatebiological information for cell culture. As a result, these materialsare unsuitable for long-term tissue culture or in vivo tissueregeneration.

On the other hand, naturally occurring scaffolds such as collagen,fibrin, alginate, hyaluronic acid, gelatin, and bacterial cellulose (BC)provide bio-functional signals and exhibit various cellularinteractions. For example, fibrin, a natural substrate of tissueremodeling (Herrick S., et al., 1999), contains several cell-signalingdomains such as a protease degradation substrate (Werb Z, 1999) andcell-adhesion domains (Herrick S., 1999). However, because suchbiological materials exhibit multiple inherent signals (e.g., regulationof cell adhesion, proliferation, cellular phenotype, matrix productionand enzyme activity), their use as scaffolds in tissue regenerationoften results in abnormal regulation of cellular events (Hubbell, 2003).Furthermore, the natural scaffolds are often much weaker afterreconstitution as compared to the strength of the original biologicalmaterial, and little control can be exercised to improve their physicalproperties.

Another drawback of natural scaffolds (e.g., collagen and fibrin) fortissue engineering is the limited control over the physical propertiesof the polymeric network. For example, reconstituted collagen undergoesfibrilogenesis and self-assembly to form an interpenetrating network ofnano-scale fibrils that loosely associate together by non-specificinteractions such as hydrogen bonding. In comparison to the highlyorganized and enzymatically cross-linked collagen fibers of the normaltissue structure, the interpenetrating network of fibrils exhibit poorphysical strength and super-physiological tissue porosity. Moreover, thespecific conformation of fibrils combined with the open pore structureof the interpenetrating network leaves the protein backbone easilyaccessibly and susceptible to freely diffusing proteases from thesurrounding host tissue or cell culture system. This often results inuncontrolled and premature deterioration of the scaffold in the presenceof cell-secreted proteases. The discrepancies in structure and functionof reconstituted protein hydrogels compared to natural urges thedevelopment of biomimetic scaffold systems for implementation in manypractical tissue engineering applications.

To date a number of techniques have been developed for the modificationand improvement of the physicochemical properties of reconstitutedprotein hydrogels which prevent them from rapid degradation. Collagenand fibrin hydrogels can be processed by freeze-drying the construct toincrease the tensile strength and modulus of the protein network.However, freeze-drying necessitates a pre-fabrication freezing stepwhich eliminates the possibility for gelation of the polymer in thepresence of cells and also eliminates the benefits of in-situpolymerization. The freeze-drying process also affects the moleculararchitecture of the polymer network, altering the nano-fiber mesh andturning it into a macro-porous sponge structure. Other techniques forimproving the physical properties of natural hydrogels while maintainingthe nano-fiber structure have been proposed based on covalentcross-links, including the use of aldehydes, carbodiimides [Park S N,Park J C, Kim H O, Song M J, Suh H. Characterization of porouscollagen/hyaluronic acid scaffold modified by1-ethyl-3-(3-dimethylaminopropyl)carbodiimide cross-linking.Biomaterials. 2002 February;23(4):1205-12], and N-hydroxysuccinimides(NHS) in the presence of amino acids [Ma L, Gao C, Mao Z, Zhou J, ShenJ. Enhanced biological stability of collagen porous scaffolds by usingamino acids as novel cross-linking bridges. Biomaterials. 2004July;25(15):2997-3004; Ma L, Gao C, Mao Z, Zhou J, Shen J.Biodegradability and cell-mediated contraction of porous collagenscaffolds: the effect of lysine as a novel crosslinking bridge. J BiomedMater Res A. 2004 Nov. 1;71(2):334-42]. All the cross-linking proceduresoffer some improvements of the physical stability of the scaffold, butdo so by introducing a toxic manufacturing step which requires extensivewashes and increases the likelihood that residual toxins in the scaffoldwill affect cellular activity.

The proteolytic degradation of protein scaffolds can also be delayed byprotecting the protein backbone of the polymer network using covalentattachment of a shielding polymers such as poly(ethylene glycol) (PEG).For example, the modification of proteins by attachment of one or morePEG chains (PEGylation) has been applied very successfully to increasingthe plasma half-life of therapeutic peptides or protein drugs [VeroneseF M. Peptide and protein PEGylation: a review of problems and solutions.Biomaterials. 2001 March;22(5):405-17]. Based on a similar rationale,PEGylation could be a good strategy for protein-based biomaterial designin as much as the PEG chains can slow down the enzymatic biodegradationof the PEGylated protein scaffold. At the same time, the PEG chains arenon-toxic, non-immunogenic, highly water soluble, and are alreadyapproved by the FDA in a number of different clinical indications(Veronese, 2005). Common proteins used in scaffold design such ascollagen and fibrin may be readily PEGylated using amine groupmodifications or thiol modifications of the protein backbone to yield aprotein-polymer conjugate. The PEG shields the protein surface fromdegrading agents by steric hindrances without blocking all the naturalbiological function of the structural protein molecule (Veronese 2005).

Recently hybrid scaffolds have been developed. A hybrid scaffoldmaterial combines the structural characteristics of the syntheticmaterial with the biofunctionality of natural material (Leach J B, etal., 2004; Leach and Schmidt, 2005). To this end, several methods ofpreparing scaffold with natural biofunctionality and physical propertiesof synthetic polymers have been proposed. Most of these “hybrid”approaches, however, fall short of producing a biomaterial with broadinherent biofunctionality and a wide range of physical properties;mainly because they employ only a single biofunctional element into thematerial design. For example, prior studies describe the preparation ofscaffolds consisting of biodegradable elements grafted into the backboneof a synthetic hydrogel network. Hydrogels were prepared from syntheticPEG which was cross-linked with short oligopeptides containing enzymaticsubstrates capable of being proteolytically degraded by cell-secretedenzymes [Lutolf et al (2003); Gobin and West (2002)]. Furthermore, toincrease the biofunctionality of such hydrogels, synthetic adhesionmotifs such as the RGD sequences [Lutolf et al (2003)] or VEGF (Seliktaret al; 2004, Zisch A H, et al, 2003; FASEB J. 17: 2260-2. Epub 2003 Oct.16) were grafted into the PEG backbone. However, the use of suchscaffolds (in which PEG is the major component) was limited by theinsufficient bio-feedback and/or long-term cellular responses which areessential for phenotypic stability.

Further attempts to increase the biofunctionality of the scaffoldsincluded the manufacture of genetically-engineered protein-likeprecursors of 100 amino acids, which contain, among other things,several protease substrates and adhesion sites (Halstenberg et al. 2002;Biomacromolecules, 3: 710-23). However, the increased protein precursorssize and the presence of thiol groups required for the PEGylationreaction complicated the purification and solubilization of theprecursors during the scaffold manufacturing process. In addition,similar to the PEG-based biosynthetic materials, thegenetically-engineered protein precursor scaffolds failed to providesufficient biofunctionality to enable long-term stability.

The present inventor has previously uncovered that biosynthetic hybridscaffolds composed of a fibrinogen backbone which is cross-linked withfunctional polyethylene glycol (PEG) side chains are excellent,biodegradable scaffolds which can be used for tissue regenerationapplications.

While reducing the present invention to practice, the present inventorhas uncovered that the above scaffold are subject to the proteolytic andhydrolytic activity of the cellular environment in the implantation sitecausing sustained release of PEGylated denatured fibrinogen degradationproducts. These PEGylated denatured fibrinogen degradation products havesimilar inductive properties of the natural fibrin degradation productswith the added advantage of the PEG modification which providesprotection from rapid clearance from the local implantation site andfrom the body.

SUMMARY OF THE INVENTION

According to one aspect of the present invention there is provided acomposition-of-matter comprising a synthetic polymer attached todenatured fibrinogen or a therapeutic portion of said denaturedfibrinogen.

According to another aspect of the present invention there is provided ascaffold comprising the composition-of-matter.

According to yet another aspect of the present invention there isprovided a hydrogel comprising the composition-of-matter.

According to further features in preferred embodiments of the inventiondescribed below, said denatured fibrinogen is fragmented denaturedfibrinogen and whereas a concentration of said units in said hydrogel isselected from a range of 0.5-35%.

According to still further features in the described preferredembodiments, modulus of elasticity of said hydrogel is in a range of0.02-0.11 kPa for 10-20% polymer.

According to still further features in the described preferredembodiments modulus of elasticity of said hydrogel is in a range of0.01-0.07 kPa for 10-20% polymer.

According to still another aspect of the present invention there isprovided a medical device comprising the composition-of-matter.

According to still further features in the described preferredembodiments the medical device is an intracorporeal device.

According to still further features in the described preferredembodiments the medical device is an extracorporeal device.

According to still further features in the described preferredembodiments the medical device is selected from the group consisting ofa prosthetic device, a pacemaker, an artificial joint, a heart valvereplacement, temporary implant, a permanent implant, a stent, a vasculargraft, an anastomotic device, a clamp, an aneurysm repair device and anembolic device.

According to an additional aspect of the present invention there isprovided a pharmaceutical composition comprising thecomposition-of-matter.

According to another aspect of the present invention there is provided amethod of treating a disorder characterized by a tissue damage, themethod comprising providing to a subject in need-thereof a compositionwhich comprises a synthetic polymer attached to denatured fibrinogen ora therapeutic portion of said fibrinogen, said composition beingformulated for releasing said therapeutic portion of said fibrinogen ina pharmacokinetically regulated manner, thereby treating the disordercharacterized by tissue damage or malformation.

According to still further features in the described preferredembodiments the composition-of-matter further comprising apharmaceutical agent.

According to still further features in the described preferredembodiments said pharmacokinetically regulated manner is immediatereleasing of said therapeutic portion of said fibrinogen.

According to still further features in the described preferredembodiments said pharmacokinetically regulated manner is sustainedreleasing of said therapeutic portion of said fibrinogen.

According to still further features in the described preferredembodiments said sustained releasing of said therapeutic portion of saidfibrinogen is between 1 week and 200 weeks.

According to still further features in the described preferredembodiments said composition is comprised in a scaffold, medical device,pharmaceutical composition or a hydrogel.

According to still further features in the described preferredembodiments said composition is formulated for local administration.

According to still further features in the described preferredembodiments said composition is formulated for systemic administration.

According to still further features in the described preferredembodiments said therapeutic portion of said denatured fibrinogen is anenzyme cleavage product of said denatured fibrinogen.

According to still further features in the described preferredembodiments said enzyme is selected from the group consisting ofplasmin, collagenase and trypsin.

According to still further features in the described preferredembodiments said therapeutic portion of said denatured fibrinogen issynthetic.

According to still further features in the described preferredembodiments said therapeutic portion of said denatured fibrinogen is asset forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7 or 8.

According to still further features in the described preferredembodiments said synthetic polymer is selected from the group consistingof polyethylene glycol (PEG), Hydroxyapatite/polycaprolactone (HA/PLC),polyglycolic acid (PGA), Poly-L-lactic acid (PLLA), Polymethylmethacrylate (PMMA), polyhydroxyalkanoate (PHA), poly-4-hydroxybutyrate(P4HB), polypropylene fumarate (PPF), polyethylene glycol-dimethacrylate(PEG-DMA), beta-tricalcium phosphate (beta-TCP) and nonbiodegradablepolytetrafluoroethylene (PTFE).

According to still further features in the described preferredembodiments said PEG is selected from the group consisting ofPEG-acrylate (PEG-Ac) and PEG-vinylsulfone (PEG-VS).

According to still further features in the described preferredembodiments said PEG-Ac is selected from the group consisting of PEG-DA,4-arm star PEG multi-Acrylate and 8-arm star PEG multi-Acrylate.

According to still further features in the described preferredembodiments said PEG-DA is a 4-kDa PEG-DA, 6-kDa PEG-DA, 10-kDa PEG-DAand/or 20-kDa PEG-DA.

According to still further features in the described preferredembodiments a molar ratio between said PEG-DA to said denaturedfibrinogen or said therapeutic portion is 2-400 to 1.

According to still further features in the described preferredembodiments a molar ratio between said PEG-DA to said fibrinogen or saidtherapeutic portion is 25 to 1.

According to still further features in the described preferredembodiments a molar ratio between said PEG-DA to said fibrinogen or saidtherapeutic portion is 75 to 1.

According to still further features in the described preferredembodiments a molar ratio between said PEG-DA to said fibrinogen or saidtherapeutic portion is 150 to 1.

According to yet an additional aspect of the present invention there isprovided an article-of-manufacturing comprising a packaging material andthe composition identified for treating a disorder characterized by atissue damage or malformation.

The present invention successfully addresses the shortcomings of thepresently known configurations by providing compositions and methods forinducing tissue regeneration.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. In case of conflict, the patentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of thepreferred embodiments of the present invention only, and are presentedin the cause of providing what is believed to be the most useful andreadily understood description of the principles and conceptual aspectsof the invention. In this regard, no attempt is made to show structuraldetails of the invention in more detail than is necessary for afundamental understanding of the invention, the description taken withthe drawings making apparent to those skilled in the art how the severalforms of the invention may be embodied in practice.

In the drawings:

FIGS. 1 a-b is a scheme depicting denaturation of purified fibrinogenmolecules. The heteromeric fibrinogen is comprised of two subunits ofthree chains each, held together by disulfide rings. Reducing thedisulfide bonds and denaturation of the fibrinogen in Urea andbeta-mercaptoethanol results in 2 sets of 3 chains of denatured protein(3 chains are shown).

FIGS. 2 a-b are protein SDS-PAGE images of denatured fibrinogenfragments prior to (FIG. 2 a) and following (FIG. 2 b) PEGylation. Note,denatured fibrinogen in the presence of PEG-OH migrates as threeindependent chains in the 50-60 kDa molecular weight range (FIG. 2 a).Cleaved fibrinogen fragments are seen below the 50 kDa molecular weightrange (FIG. 2 b).

FIGS. 3 a-b are schemes showing PEGylated fibrinogen (whole, FIG. 3 a)and fibrinogen degradation products (cleaved, FIG. 3 b). The boundpolymer can be monofunctional, difunctional (shown), or multifunctional.The remaining reactive groups on the polymer can be used to covalentlybind the denatured fibrinogen degradation products or whole denaturedfibrinogen into a 3-D matrix for local implantation;

FIGS. 4 a-b show the creation of a matrix from denatured PEGylatedfibrinogen showing the molecular architecture schematically illustratedin FIG. 4 a and the macroscopic appearance of FIG. 4 b;

FIGS. 5 a-d depict the in vitro degradation kinetics of denaturedPEGylated fibrinogen precursor (whole) into degradation products bytrypsin (FIGS. 5 a-b) and collagenase (FIGS. 5 c-d).

FIGS. 6 a-j are photographs depicting smooth muscle cells migration fromsmooth muscle tissue into PEG-Fibrinogen containing hydrogel matrix.Smooth muscle tissue constructs were encapsulated by PEG-fibrinogenhydrogel and visualized by phase-contrast microscopy. Cellular invasionfrom the dense tissue (opaque) into the hydrogels (transparent) was seenwithin several hours following hydrogel casting and throughout theexperiment (FIGS. 6 a-c). Macroscopically, the tissue construct (arrow)was entrapped within the PEG-fibrinogen hydrogel (FIGS. 6 d-e). Of note,the hydrogel maintained its structural stability even following one weekin culture (FIG. 6 f). The degradation kinetics and invasion kineticswere affected by the amount relative composition of PEG-DA and denaturedfibrinogen in hydrogel; the cells became more polarized and orientedradially outward from the tissue construct as they invadedPEG-fibrinogen matrix containing higher concentrations of 10-kDa PEG-DA(FIG. 6 g—0%; FIG. 6 h—0.5%; FIG. 6 i—1.0%; FIG. 6 j—2.0%). Phasecontrast micrographs were imaged at 40× magnification (scale bar=250μm);

FIGS. 7 a-d are illustrate cellular invasion resultant of thechemotactic activity of degrading denatured PEGylated fibrinogenhydrogels. FIG. 7 a shows the invasion kinetics data during one-weekculture of smooth muscle cells invading into 10-kDa PEGylated fibrinogenhydrogels made with additional PEG-DA. FIG. 7 b shows the summary ofinvasion data for 6-kDa and 10-kDa PEGylated fibrinogen hydrogels duringone week. FIG. 7 c-d depict stem cell and cartilage cell invasion intohydrogel materials releasing denatured PEGylated fibrinogen degradationproducts. Each image shows an in vitro culture of a three-dimensionallyentrapped living, natural tissue explants within a dense hydrogelmatrix. FIG. 7 c shows human embryoid bodies (EB) cultured for 8 daysdemonstrating embryonic stem cell sprouting from the EB into the densegel matrix. FIG. 7 d shows cartilage explants (CE) cultured for 14 daysexhibiting massive cell invasion from the native tissue into theencapsulating gel matrix.

FIGS. 8 a-d are photographs depicting the generation of a site-specificdefect in the rat's tibia. FIG. 8 a—The mid-portion of the right tibiawas exposed from the anterior medial side by longitudinal incision; FIG.8 b—An external fixation device was placed proximal and distal to themid-section of the tibia; FIG. 8 c—A 7-mm gap was excised in the portionbetween the proximal and distal needles of the fixation device; and FIG.8 d—A PEG-fibrinogen containing plug was inserted into the defect site.

FIGS. 9 a-c are photomicrographs depicting longitudinal sections ofintermediate-degrading hydrogel-treated tibial defects 5 Weekspost-operation. The extent of regenerated bone in the site-specificdefect ranged from partial (FIGS. 9 a-b) to total bridging (FIG. 9 c) ofthe defect osteotomies (ost), and highly depended on the erosion patternof the hydrogel material (Gel). Remnants of the gel gave way toregenerated bone (dashed line), having typical lamellar-fibred patternof mature osseous trabeculae and fatty marrow (FM). The sections werestained with hematoxylin and eosin (H&E);

FIGS. 10 a-b are photomicrographs depicting newly formed subperiostealand endosteoal bone shown with partially degraded hydrogel in alongitudinal section of an intermediate-degrading treatment. FIG. 10a—The osseous trabeculae, which connect with one another, are rimmed byactive cuboidal osteoblasts. The intertrabecular spaces are occupied bya fatty marrow (FM), which well extends into the site of the defect fromthe aspect of the medial osteotomy (ost). A cartilaginous cap (arrows)at the medial end of the front of regenerated bone is seen with isletsof hypertrophic chondrocytes. The cap is enclosed by a thin layer ofperichondrium-like fibrous tissue. Fibro-fatty tissue (FT) is present inbetween the degraded hydrogel and the regenerated bone. FIG. 10 b—Thisfield (taken at higher magnification) displays endochondrol ossification(ECO) in the cartilaginous region. The sections are stained withhematoxylin and eosin (H&E);

FIGS. 11 a-b are photomicrographs depicting cellular response to thePEG-fibrinogen containing hydrogel implant. FIG. 11 a shows a serpentinegranulation tissue at the eroding front of the hydrogel (solid arrows)with an adjacent nonspecific chronic inflammatory infiltrate, which isprimarily composed of lymphocytes (LR) and is accompanied by newlyformed bone (NB). In certain areas of the tissue-material interface, theresponse is limited to a minor chronic nonspecific inflammatory reaction(dashed arrow). The eosinophilic hydrogel is lightly stained and showsno cellular infiltration beyond the eroding borders of the dense matrix(gel). FIG. 11 b is a high magnification micrograph showing thepallisading granulation tissue. Of note is the minor macrophagicreaction (MR).

FIGS. 12 a-d are pictures showing implantation of denatured fibrinogenimplant in oseteochondral defects. FIG. 12 a shows gross appearance offour osteochondral defects, 6-mm in diameter situated in the femoralcondyle in the right-stifle joint. FIG. 12 b shows the PEGylateddenatured fibrinogen implant polymerized in situ in the osteochondraldefect. FIGS. 12 c-d show an operating room scene where the surgeoninjects the polymer solution into the defect site and polymerizes thesolution in situ using a UV light source and a fiber-optic light guide.

FIGS. 13 a-d are pictures showing the gross appearance of osteochondroldefects on the day of operation (FIGS. 13 a-b) and 4 months, following,during tissue harvest. A newly formed articular (hyaline) cartilagesurface is seen in treated defects (1, 2, 5) whereas the empty controldefect appears dramatically different. FIG. 13 a—patellar notch day ofoperation; FIG. 13 b—femoral condyle day of operation; FIG. 13c—patellar notch 4 months post-operative; FIG. 13 d—femoral condyle 4months post-operative.

FIGS. 14 a-d are longitudinal histological sections of the 6-mmosteochondral defects 4 months following implantation. The defects areeither left empty (FIG. 14 a empty control) or treated with hydrogels ofdenatured PEGylated fibrinogen (FIGS. 14 b-d) with differentconcentrations of PEG and fibrinogen in each treatment. Histology isshown for treatments with (FIG. 14 b) 150:1 molar ratio of PEG tofibrinogen, (FIG. 14 c) 200:1 molar ratio of PEG to fibrinogen, and(FIG. 14 d) 350:1 molar ratio PEG to fibrinogen in the defect site. Alltreated defects showed impressive regeneration of both cartilage andbone surrounding the eroding PEGylated fibrinogen implant. In all thehistological sections of the treated defects, the acellular gel is seenand marked in the middle of the H&E stained section.

FIGS. 15 a-d are longitudinal histological sections of the 6-mmosteochondral defects 4 months following implantation stained for Type Icollagen using monoclonal antibodies. The defects are either left empty(FIG. 15 a empty control) or treated with hydrogels of denaturedPEGylated fibrinogen (FIGS. 15 b-d) with different concentrations of PEGand fibrinogen in each treatment. Histology is shown for treatments with(FIG. 15 b) 150:1 molar ratio of PEG to fibrinogen, (FIG. 15 c) 200:1molar ratio of PEG to fibrinogen, and (FIG. 15 d) 350:1 molar ratio PEGto fibrinogen in the defect site. All treated defects showed new bonestaining positive for Type I collagen (brown color).

FIGS. 16 a-d are longitudinal histological sections of the 6-mmosteochondral defects 4 months following implantation stained forproteoglycans using safranin-o stain. The defects are either left empty(FIG. 16 a empty control) or treated with hydrogels of denaturedPEGylated fibrinogen (FIGS. 16 b-d) with different concentrations of PEGand fibrinogen in each treatment. Histology is shown for treatments with(FIG. 16 b) 150:1 molar ratio of PEG to fibrinogen, (FIG. 16 c) 200:1molar ratio of PEG to fibrinogen, and (FIG. 16 d) 350:1 molar ratio PEGto fibrinogen in the defect site. All treated defects showed newcartilage staining positive for proteoglycans (red color), compared toempty control which does not stain positive. The proteoglycan stainingusing safranin-o (red stain is positive) confirms the presence ofglycosaminoglycans (GAG) in the newly formed cartilage surface.

FIGS. 17 a-d are longitudinal histological sections of the 6-mmosteochondral defects 4 months following implantation stained for TypeII collagen using monoclonal antibodies. The defects are either leftempty (FIG. 17 a empty control) or treated with hydrogels of denaturedPEGylated fibrinogen (FIGS. 17 b-d) with different concentrations of PEGand fibrinogen in each treatment. Histology is shown for treatments with(FIG. 17 b) 150:1 molar ratio of PEG to fibrinogen, (FIG. 17 c) 200:1molar ratio of PEG to fibrinogen, and (FIG. 17 d) 350:1 molar ratio PEGto fibrinogen in the defect site. All treated defects showed newcartilage staining positive for collagen type II (brown color), comparedto new bone which does not stain positive.

FIGS. 18 a-b are histological sections of osteochondral defects treatedwith PEGylated fibrinogen implants shown at the implant-tissue interfaceafter 4 months. FIG. 18 a shows a thin layer of an inflammatoryinfiltrate surrounding the eroding implant. A higher magnification imageof this interface (FIG. 18 b) shows the implant, the inflammatoryresponse and the newly formed cartilage and bone cells.

FIGS. 19 a-d are high magnification images of articular cartilage repairshowing the newly formed hyaline cartilage in the PEGylated fibrinogentreated defects with (FIGS. 19 a-b) 150:1 molar ratio PEG to fibrinogenand (FIGS. 19 c-d) 350:1 molar ratio PEG to fibrinogen.

FIGS. 20 a-f show the integration of the defect site with the newlyformed hyaline cartilage in the sheep osteochondral defect model. FIG.20 a shows the original dimensions of the defect site which is marked byshort arrows. FIGS. 20 c-f show longitudinal high magnification imagesof the entire length of the newly formed articular cartilage highlightthe extent and quality of healing. Integration between the new and oldcartilage is marked by long arrows in (FIG. 20 b) and (FIG. 20 f).

FIGS. 21 a-c are photographs showing the encapsulation of a singledorsal root ganglion (DRG) into a PEGylated fibrinogen hydrogel. FIG. 21a illustrates that the DRG (arrow) is roughly 0.5 mm in diameter and issituated in the center of a 10 mm diameter PEGylated fibrinogenhydrogel. Radial outgrowth is measured from the outer boundary of theopaque DRG into the transparent hydrogel. The same constructs is shownfrom the top view (FIG. 21 b) and the side view (FIG. 21 c).

FIGS. 22 a-d are photomicrographs showing outgrowth and cellularinvasion characteristics of DRGs in PEGylated fibrinogen 3-D hydrogelconstructs. Phase-contrast micrographs (FIGS. 22 a and 22 b) show thethree-dimensional outgrowth of neurites (arrow) and glial cells(arrowhead) extending from the DRG (D) into the transparent PEGylatedfibrinogen hydrogel construct (P) following two days in culture.Histological sections stained with H&E (FIGS. 22 c and 22 d) of the DRG(dark) in the PEGylated fibrinogen construct (light) show neurite(arrow) and non-neuronal cells (arrowhead) invading the hydrogels afterfour days in culture. Note: high magnification images (FIGS. 22 b and 22d) are expanded regions from the lower magnification micrographs (FIGS.22 a and 22 c); in all images the scale bar=100 μm.

FIGS. 23 a-f are fluorescent microscope images of DRGs encapsulated inPEGylated fibrinogen constructs confirming the presence of both neuritesand schwann cells. Cross sections of DRG constructs were cultured forfour days and fluorescently triple-labeled with βIII-tubulin (neuritemarker, FIGS. 23 a and 23 d), s100 (Schwann cell marker, FIGS. 23 b and23 e), and DAPI counter-stain (nuclei, blue). The merged micrographs(FIGS. 23 c and 23 f) show the three-dimensional invasion of neuritesfrom the DRG into the hydrogel construct, with Schwann cells associatedvery closely with the neurite extensions (scale bar=50 μm).

FIGS. 24 a-d are fluorescent microscope images and graphs showing thatboth free soluble and enmeshed nerve growth factor (FS-NGF and EN-NGF)promote 3-D neurite outgrowth from encapsulated DRGs into the hydrogels.Sections of DRG constructs following four days were immunofluorescentlylabeled for βIII-tubulin (neurites, red), s100 (Schwann cells, green)and DAPI nuclear stain (blue) to characterize the invasion into thehydrogels containing FS-NGF (FIG. 24 a), EN-NGF (FIG. 24 b), or no NGF(NO-NGF; FIG. 24 c). Absence of NGF (NO-NGF) did not encourage outgrowthof neurites but supported moderate outgrowth of Schwann cells.Treatments with free soluble or enmeshed NGF exhibited impressiveoutgrowth of both neurites and Schwann cells (scale bar=50 μm). FIG. 24d is a line graph illustrating that the average neurite extension length(±standard error) in free soluble NGF treatments (FS-NGF) and enmeshedNGF treatments (EN-NGF) is not significantly different between the twotreatments (P>0.35, n=6).

FIGS. 25 a-u are phase contrast micrographs and graphs showing thatPEG-fibrinogen composition controls neurite invasion and outgrowth. Thehydrogel composition is varied using different amounts of PEG andfibrinogen during assembly, including 30:1, 60:1, 120:1, and 180:1(PEG:Fibrinogen). FIGS. 25 a-p are phase contrast micrographs showingthe profound impact of additional PEG on the 3-D outgrowth morphologyfrom the DRG following four days (scale bar=200 μm). FIGS. 25 q-t arehigh magnification images showing the relative outgrowth of neurites andglial cells into hydrogels having different compositions (scale bar=200μm). FIG. 25 u is a line graph showing that the average neuriteextension length (±standard error) in each treatment, as measureddirectly from the images, shows no significant difference between the30:1 and 60:1 treatments (P>0.50, n=9), and a significant impediment tooutgrowth in the 120:1 and 180:1 treatments (p<0.01, n=9).

FIGS. 26 a-c are phase contrast micrographs showing DRG outgrowth intohydrogels made from PEG-DA and PEG-fibrinogen. Constructs were preparedwith 10% PEG-DA gels (w/v) without fibrinogen (FIG. 26 a) and comparedwith PEG-fibrinogen constructs (FIG. 26 b). Neurite extensions werebarely visible following three days in PEG-DA construct compared toextensive invasion seen in PEGylated fibrinogen constructs after threedays. Neuronal invasion into PEGylated fibrinogen hydrogels waseliminated in the absence of NGF (FIG. 26 c), although other cells typeswere observed in the hydrogel following three days in culture. In allimages the scale bar=200 μm.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is of novel compositions which can be used fortreating tissue damage, loss or malformation.

The principles and operation of the present invention may be betterunderstood with reference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details set forth in the following description or exemplified bythe Examples. The invention is capable of other embodiments or of beingpracticed or carried out in various ways. Also, it is to be understoodthat the phraseology and terminology employed herein is for the purposeof description and should not be regarded as limiting.

Material technologies have long been harnessed for tissue regenerationapplications. Biomaterials used in tissue regeneration are composed ofnatural or biological materials, synthetic materials, and biosyntheticcomposite or hybrid materials. Natural materials such as collagen andfibrinogen, may exert uncontrolled cell signaling and may introduceuncertainty in the regulated communication between the biomaterial andcells. Moreover, the inherent structural characteristics of biologicalmaterials are not easily altered, thus limiting control over physicalcues received by the surrounding cells and tissues. Synthetic scaffoldsare generated mostly from biologically inactive (inert) materials thatare easily manipulated, rendering these ideal for regulating thephysical interactions between cells and biomaterial at thebiomaterial-tissue interface. However, synthetic scaffolds mayinadvertently participate in random biological signaling caused byabsorption of serum proteins, for example, and subsequent activation ofcells through contact with these proteins. Biosynthetic compositematerials are a complementary blend of natural and synthetic materialsdesigned to regulate between the physical and biological cell signalingof the scaffold.

The present inventor has previously uncovered that biosynthetic hybridscaffolds composed of a fibrinogen backbone which is cross-linked withfunctional polyethylene glycol (PEG) side chains are excellent,biodegradable scaffolds which can be used for tissue regenerationapplications (see WO2005/055800).

While reducing the present invention to practice, the present inventorhas uncovered that once implanted the above scaffolds are subject to theproteolytic and hydrolytic activity of the cellular environment causingsustained release of PEGylated denatured fibrinogen degradationproducts. These PEGylated denatured fibrinogen degradation products havesimilar inductive properties of the natural fibrin degradation productswith the added advantage of the PEG modification (PEGylation) and whichprovides protection from rapid clearance from the local implantationsite and from the body. Such compositions may be formulated forreleasing these therapeutically active fibrin fragments in apharmacokinetically regulated manner, allowing the treatment of a myriadof diseases associated with tissue damage, loss or malformation.

Thus, as is illustrated hereinbelow and in the Examples section whichfollows, the present inventor designed a biosynthetic hybrid fibrinogenprecursor by subjecting cleaved or whole denatured fibrinogen toPEGylation, possibly generating a three dimensional (3D) matrixtherefrom (see Example 1). The present inventor further showed thatdegradation products released from the above scaffold have a chemotacticactivity as shown in an in vitro tissue culture assay (see Example 3).The fibrinogen precursors were formulated of various PEG:denaturedfibrinogen ratios for controlling release of the active products at thesite of implantation. Such formulations were shown effective in bone andcartilage regeneration in animal models of injury (see Examples 4 and5).

Thus, according to the present invention there is provided a method oftreating a disorder characterized by tissue damage. The methodcomprising providing to a subject-in-need-thereof a therapeuticallyeffective amount of a composition which comprises a synthetic polymerattached to fibrinogen or a therapeutic portion of the fibrinogen, thecomposition being formulated for releasing the therapeutic portion ofthe fibrinogen in a pharmacokinetically regulated manner, therebytreating the subject having the disorder characterized by tissue damage.

As used herein the term “subject” refers to an animal subject, such as amammal, preferably a human subject.

As used herein the term “treating” refers to alleviating or diminishinga symptom associated with a disorder. Preferably, treating cures, e.g.,substantially eliminates, the symptoms associated with the disorder.

As used herein the phrase “disorder characterized by a tissue damage”refers to a disorder, disease or condition which is caused by orassociated with a non functioning tissue (i.e., cancerous orpre-cancerous tissue, wounded tissue, broken tissue, fractured tissue,fibrotic tissue, or ischemic tissue); and/or tissue loss (reduced amountof functioning tissue) such as following a trauma, an injury or abnormaldevelopment (i.e., malformation,—structural defect that occursinfrequently such as due to abnormal development which require tissueregeneration). Preferably the tissue is a functional tissue such as abone tissue, a cartilage tissue, a tendon tissue, ligament, a cardiactissue, a nerve tissue, or a muscle tissue.

Examples of disorders characterized by a tissue damage include, but arenot limited to, cartilage damage (articular, mandibular), bone cancer,osteoporosis, bone fracture or deficiency, primary or secondaryhyperparathyroidism, osteoarthritis, periodontal disease or defect, anosteolytic bone disease, post-plastic surgery, post-orthopedicimplantation, post-dental implantation, cardiac ischemia, muscleatrophy, and nerve degeneration.

As mentioned in the above subject is administered with a compositionwhich comprises a synthetic polymer attached to the denatured fibrinogenor a therapeutic portion of the denatured fibrinogen.

As used herein “denatured fibrinogen” refers to each of the 6 chainscomposing fibrinogen which is formed by any mode of specific (disulfide)and non-specific (hydrogen bond) denaturation such as by thermaldenaturation, freeze-drying, alkali denaturation and chelator (e.g.,EGTA) mediated denaturation [Haddeland (1995) Thromb. Res. 77:329-36].Denatured fibrinogen may comprise a whole fibrinogen chain or atherapeutic portion thereof.

As used herein “a therapeutic portion of fibrinogen” refers to afibrinogen portion which is sufficient to mediate a fibrinogen activity,such as cell proliferation, angiogenesis or anti-inflammatory activity.

The amino acid length of the therapeutic portion of fibrinogen may vary.Such as at least 3 amino acids long (e.g., 3-20, 3-50, 3-100, 3-200,3-300, 3-400, 3-500 amino acids).

Examples of therapeutic portions of fibrinogen are provided in SEQ IDNOs. 1-8 (see e.g., U.S. Pat. Nos. 5,427,918, 5,473,051, 5,919,754; U.S.Pat. Appl. Nos. 20030109431, 20050020809, 20040126758, 20040029157).

Therapeutic portions of fibrinogen may be generated by enzymaticcleavage of fibrinogen using one or more enzymes which cleavefibrinogen. Examples of such enzymes include, but are not limited to,plasmin, collagenase and trypsin. See example 2 of the Examples sectionwhich follows.

Alternatively, therapeutic portions of the denatured fibrinogen may begenerated by a proteolytic chemical such as cyanogen bromide and2-nitro-5-thiocyanobenzoate.

Yet alternatively, therapeutic portions of denatured fibrinogen may begenerated using synthetic or recombinant techniques which are well knownin the art.

Synthetic peptides can be prepared by classical methods known in theart, for example, by using standard solid phase techniques. The standardmethods include exclusive solid phase synthesis, partial solid phasesynthesis methods, fragment condensation, classical solution synthesis,and even by recombinant DNA technology. See, e.g., Merrifield, J. Am.Chem. Soc., 85:2149 (1963), incorporated herein by reference. Solidphase peptide synthesis procedures are well known in the art and furtherdescribed by John Morrow Stewart and Janis Dillaha Young, Solid PhasePeptide Syntheses (2nd Ed., Pierce Chemical Company, 1984).

Regardless of the method employed, once therapeutic portions aregenerated they may be used in a batch which comprises a mixture oftherapeutic portions with or without inactive portions, oralternatively, isolated and purified (e.g., medical purity e.g., over95% purity). In this regard, it will be appreciated that while somemolecules may not have a therapeutic effect, such molecules can stillserve in combination with those having a therapeutic effect, as part ofthe carrier in a formulation, such as further described herein below.Essaying therapeutic activity of fibrinogen products may be effectedusing methods which are well known in the art such as cell proliferationor migration assay, described in length in the Examples section whichfollows.

Thus, therapeutic products can be purified by preparative highperformance liquid chromatography [Creighton T. (1983) Proteins,structures and molecular principles. WH Freeman and Co. N.Y.], gelelectrophoresis and the composition of which can be confirmed via aminoacid sequencing.

As mentioned, denatured fibrinogen or portions thereof are attached(i.e., covalently attached) to a synthetic polymer. Methods ofcovalently attaching synthetic polymers to fibrinogen are well known inthe art and described in Example 1 of the Examples section.

Non-limiting examples for synthetic polymers which can be used alongwith the present invention include polyethylene glycol (PEG) (averageMw. 200; P3015, SIGMA), Hydroxyapatite/polycaprolactone (HA/PLC) [Choi,D., et al., 2004, Materials Research Bulletin, 39: 417-432; Azevedo M C,et al., 2003, J. Mater Sci. Mater. Med. 14(2): 103-7], polyglycolic acid(PGA) [Nakamura T, et al., 2004, Brain Res. 1027(1-2): 18-29],Poly-L-lactic acid (PLLA) [Ma Z, et al., 2005, Biomaterials. 26(11):1253-9], Polymethyl methacrylate (PMMA) [average Mw 93,000, Aldrich Cat.No. 370037; Li C, et al., 2004, J. Mater. Sci. Mater. Med. 15(1): 85-9],polyhydroxyalkanoate (PHA) [Zinn M, et al., 2001, Adv. Drug Deliv. Rev.53(1): 5-21; Sudesh K., 2004, Med. J. Malaysia. 59 Suppl B: 55-6],poly-4-hydroxybutyrate (P4HB) [Dvorin E L et al., 2003, Tissue Eng.9(3): 487-93], polypropylene fumarate (PPF) [Dean D, et al., 2003,Tissue Eng. 9(3): 495-504; He S, et al., 2000, Biomaterials, 21(23):2389-94], polyethylene glycol-dimethacrylate (PEG-DMA) [Oral E andPeppas N A J, 2004, Biomed. Mater. Res. 68A(3): 439-47], beta-tricalciumphosphate (beta-TCP) [Dong J, et al., 2002, Biomaterials, 23(23):4493-502], and nonbiodegradable polytetrafluoroethylene (PTFE) [JerniganT W, et al., 2004. Ann. Surg. 239(5): 733-8; discussion 738-40].

According to a presently preferred embodiment of the present inventionthe synthetic polymer used by the present invention is PEG. The PEGmolecule used by the present invention can be linearized or branched(i.e., 2-arm, 4-arm, and 8-arm PEG) and can be of any molecular weight,e.g., 4 kDa, 6 kDa and 20 kDa for linearized or 2-arm PEG, 14 kDa and 20kDa for 4-arm PEG, and 14 kDa and 20 kDa for 8-arm PEG and combinationthereof.

According to a presently known preferred embodiment of the presentinvention, a 6-14 kDa PEG-diacrylate is used.

It will be appreciated that the OH-termini of the PEG molecule can bereacted with a chemical group such as acrylate (Ac) or vinylsulfone (VS)which turn the PEG molecule into a functionalized PEG, i.e., PEG-Ac orPEG-VS. Preferably, the PEG molecule used by the present invention isPEG-Ac. Alternatively; the PEG can be modified with a pyridildisulphidegroup, a maleimide group, or a iodo acetamide group. Other functionalgroups include carbonates such as benzotriazolyl carbonate andsuccinimidyl carbonate [see review by Veronese F and Paust G, DrugDiscovery Reviews, vol 10 (21), November 2005].

Methods of preparing functionalized PEG molecules are known in the arts.For example, PEG-VS can be prepared under argon by reacting adichloromethane (DCM) solution of the PEG-OH with NaH and then withdi-vinylsulfone (molar ratios: OH 1: NaH 5: divinyl sulfone 50, at 0.2gram PEG/mL DCM). PEG-Ac is made under argon by reacting a DCM solutionof the PEG-OH with acryloyl chloride and triethylamine (molar ratios: OH1: acryloyl chloride 1.5: triethylamine 2, at 0.2 gram PEG/mL DCM),essentially as described in Example 1 of the Examples section whichfollows.

It will be appreciated that such chemical groups can be attached tolinearized, 2-arm, 4-arm, or 8-arm PEG molecules.

Preferably, the PEG-Ac used by the present invention is PEG-DA, 4-armstar PEG multi-Acrylate and/or 8-arm star PEG multi-Acrylate.

As is shown in Example 1 of the Examples section which follows thepresent inventor used various isoforms of PEG-diacrylate (PEG-DA) toprepare functionalized PEG molecules.

As mentioned hereinabove, the above precursor composition (i.e.,synthetic polymer attached to denatured fibrinogen or a therapeuticportion of same) is formulated for releasing the therapeutic portion ofthe fibrinogen in a pharmacokinetically regulated manner. One ofordinary skill in the art will choose the formulation according to theintended use.

The pharmacokinetic of the therapeutic portion of fibrinogen (i.e.,action of the therapeutic portion in the body over a period of time) maybe governed by the following exemplary parameters: syntheticpolymer:fibrinogen ratio, 3-D matrix formation, use of whole denaturedfibrinogen or therapeutic portions of same, mode of administration. Eachparameter can affect the biological activity of the release fibrinogenfragment, the release kinetics, the clearance rate from the body, andthe immunogenic response of the therapeutic factor.

The following Table 1 provides exemplary formulations for variousindications. TABLE 1 Weeks PEG/denatured following Uses/ Mode offibrinogen Release rate administration indications administrationFormulation molar ratio Slow 10-150 Bone, cartilage, Implantation, insitu Hydrogel 100:1-1000:1 cardiac, neural polymerization Intermediate2-10 Bone, Muscle Implantation, in situ Hydrogel 20:1-100:1polymerization Fast 1-2  Treatment of local In situ polymerizationCross-linked 10:1-20:1  infection in skin, bone, interstitium Immediate1 Non-critical size In situ injection Non-cross linked  2:1-1000:1injuries composition

The following describes the different parameters affectingpharmacokinetics:

3-D matrix formation—Cross-linking the polymer-protein precursormolecules of the present invention may affect pharmacokinetics of thetherapeutic portion. Such cross-linking can be performed in vitro, exvivo and/or in vivo.

Cross-linking is performed by subjecting the precursor molecules to afree-radical polymerization reaction (i.e., a cross-linking reaction).Methods of cross-linking polymers are known in the art, and include forexample, cross-linking via photoinitiation (in the presence of anappropriate light, e.g., 365 nm), chemical cross-linking [in thepresence of a free-radical donor] and/or heating [at the appropriatetemperatures. Preferably, cross-linking according to the presentinvention is effected by photoinitiation.

Photoinitiation can take place using a photoinitiation agent (i.e.,photoinitiator) such as bis(2,4,6-trimethylbenzoyl)phenylphosphine oxide(BAPO) (Fisher J P et al., 2001; J. Biomater. Sci. Polym. Ed. 12:673-87), 2,2-dimethoxy-2-phenylacetophenone (DMPA) (Witte R P et al.,2004; J. Biomed. Mater. Res. 71A(3): 508-18), camphorquinone (CQ),1-phenyl-1,2-propanedione (PPD) (Park Y J et al., 1999, Dent. Mater.15(2): 120-7; Gamez E, et al., 2003, Cell Transplant. 12(5): 481-90),the organometallic complex Cp′Pt(CH(3))(3) (Cp′=eta(5)-C(5)H(4)CH(3))(Jakubek V, and Lees A J, 2004; Inorg. Chem. 43(22): 6869-71),2-hydroxy-1-[4-(hydroxyethoxy)phenyl]-2-methyl-1-propanone (Irgacure2959) (Williams C G, et al., 2005; Biomaterials. 26(11): 1211-8),dimethylaminoethyl methacrylate (DMAEMA) (Priyawan R, et al., 1997; J.Mater. Sci. Mater. Med. 8(7): 461-4), 2,2-dimethoxy-2-phenylacetophenone(Lee Y M et al., 1997; J. Mater. Sci. Mater. Med. 8(9): 537-41),benzophenone (BP) (Wang Y and Yang W. 2004; Langmuir. 20(15): 6225-31),flavin (Sun G, and Anderson V E. 2004; Electrophoresis, 25(7-8):959-65).

The photoinitiation reaction can be performed using a variety ofwave-lengths including UV (190-365 nm) wavelengths, and visible light(400-1100 nm) and at various light intensities. It will be appreciatedthat for ex vivo or in vivo applications, the photoinitiator andwavelengths used are preferably non-toxic and/or non-hazardous.

For example, the PEG-fibrinogen precursor molecule can be cross-linkedby photoinitiation in the presence of Irgacure™ 2959 and a non-toxic UVlight illumination (e.g., 5 minutes at 365 nm wavelength, 4-5 mWatts/cm²intensity).

It will be appreciated that although PEGylated fibrinogen molecules ofthe present invention are capable of being cross-linked without theaddition of a cross-linking molecule, cross-linking according to thepresent invention can also utilize a molecule capable of cross-linkingthe polymer-protein precursors. Such cross-linking molecules can be forexamples, PEG, PEG-DA, PEG multi-Acrylate, and/or PEG-VS.

Cross-linking may be effected such that the polymer-protein precursorsof the present invention are solubilized in a water-based solution andsuch solutions are further subjected to cross-linking (e.g., usingphotoinitiation) to form a hydrogel scaffold. The hydrogel is subjectedto the in vivo eroding conditions and releases the therapeutic portionsof the denatured fibrinogen.

For example, a PEG-fibrinogen hydrogel was formed by mixing thePEGylated fibrinogen precursor molecules with the photoinitiation agentin the presence or absence of PEG-DA and exposing such a mixture to UVlight. Briefly, the PEGylated fibrinogen precursors were solubilized in1-ml of 50 mM PBS, pH 7.4 and 25° C. to achieve a final concentration of10, 15, or 20% polymer-protein (w/v). The precursor solution alsocontained a PEG-DA cross-linking constituent at a molar ratio of 1:2PEG-DA to functional groups on the PEGylated fibrinogen. The precursorsolution was mixed with 10 μl of Irgacure™ 2959 photoinitiator solution(Ciba Specialty Chemicals, Tarrytown, N.Y.) in 70% ethanol (100 mg/ml)and centrifuged for 5 min at 14,000 RPM. The solution was then placedinto Teflon tubes (5-mm diameter and 20-mm long) and polymerized underUV light (365 nm, 4-5 mW/cm²) for 15 minutes according to publishedprotocols (Lum L Y et al., 2003).

According to preferred embodiments of the present invention the hydrogelcan be generated from PEGylated whole denatured fibrinogen or PEGylatedfragmented fibrinogen (therapeutic portions). Generally, the molecularweight and length of the grafted PEG affects the degree of solubility ofthe PEGylated protein, i.e., higher length and/or molecular weight ofPEG results in increased solubility of PEGylated protein. It will beappreciated that solubility of the PEGylated protein is also affected bythe presence of whole or cleaved fibrinogen. Preferably, theconcentration of the precursor molecules in the hydrogel is between 0.5to 35%, more preferably, when PEGylated whole fibrinogen is used, theconcentration of the precursor molecules in the hydrogel is between 0.5to 5% (depending on the MW and length of the grafted PEG used toPEGylate the protein) and when PEGylated fragmented fibrinogen is used,the concentration of the precursor molecules in the hydrogel is between5-35% (depending on the MW and length of PEG used to PEGylate theprotein).

Synthetic polymer:fibrinogen ratio—the molar ratio between the syntheticpolymer (e.g., PEG) and fibrinogen of the present invention may affectpharmacokinetics of the composition. Thus, excess of the syntheticpolymer would lead to binding of the polymer functional groups (e.g.,PEG-DA) to all potential binding sites on the fibrinogen and, such thatwhen cross-linked would result in a denser mesh with slower releasepattern. On the other hand, binding of only two molecules of thesynthetic polymer to each molecule of the protein (i.e., a 2:1 molarratio) would result in fewer cross-linking sites and higherbiodegradability of the scaffold. Thus, a higher molar ratio (i.e.,excess of polymer) is expected to result in less biodegradability due topotential masking of protein degradation sites. Those of skills in theart are capable of adjusting the molar ratio between the syntheticpolymer and the protein to obtain the desired formulation with theoptimal physical and biological characteristics.

For example, since each fibrinogen molecule includes 29-31 potentialsites which can bind to PEG, the PEG-fibrinogen precursor molecule canbe prepared using a wide range of molar ratios. Preferably, the molarratio used by the present invention is 2-400 (PEG) to 1 (fibrinogen),more preferably, the molar ratio is 30-300 (PEG) to 1 (fibrinogen), morepreferably, the molar ratio is 100-200 (PEG) to 1 (fibrinogen), mostpreferably, the molar ratio is 130-160 (PEG) to 1 (fibrinogen). As isshown in Example 1 of the Examples section which follows, one preferablemolar ratio between PEG-DA and fibrinogen is 145 (PEG) to 1(fibrinogen). In the case where the molar ratio is greater than 29-31(PEG) to 1 (fibrinogen), some of the PEG can be indirectly bound to thefibrinogen through fibrinogen-bound PEG molecules.

The fibrinogen used by the present invention can be whole denaturedfibrinogen (i.e., un-cleaved) or fragmented fibrinogen, which can beobtained using, for example, CNBr cleavage.

Fibrinogen can be readily purified from human blood plasma usingstandard protein purification techniques. Purified components may besubject to anti-viral treatments. Heat-treatment and solvent/detergenttreatments are both commonly used in the production of fibrinogen.Fibrinogen used in accordance with the present invention is preferablypure though other components may be present. Thus products may alsocontain tranexamic acid, aprotinin or factor XIII. Fibrinogen iscommercially available from Baxter and Omrix.

Routes of administration—Pharmacokinetics of the compositions of thepresent invention may be affected by the mode of administration. Hencepharmaceutical compositions which comprise the therapeutic portions offibrinogen as an active ingredient or in a pro-drug form may beformulated for local or systemic administration.

Techniques for formulation and administration of drugs may be found inthe latest edition of “Remington's Pharmaceutical Sciences,” MackPublishing Co., Easton, Pa., which is herein fully incorporated byreference.

Suitable routes of administration may, for example, include oral,rectal, transmucosal, especially transnasal, intestinal, or parenteraldelivery, including intramuscular, subcutaneous, and intramedullaryinjections, as well as intrathecal, direct intraventricular,intravenous, inrtaperitoneal, intranasal, or intraocular injections.

Alternately, one may administer the pharmaceutical composition in alocal rather than systemic manner, for example, via injection of thepharmaceutical composition directly into a tissue region of a patient.

Compositions of the present invention may be manufactured by processeswell known in the art, e.g., by means of conventional mixing,dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping, or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in conventional manner using one ormore physiologically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active ingredients intopreparations that can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the active ingredients of the pharmaceutical compositionmay be formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hank's solution, Ringer's solution, orphysiological salt buffer. For transmucosal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art.

Pharmaceutical compositions suitable for use in the context of thepresent invention include compositions wherein the active ingredientsare contained in an amount effective to achieve the intended purpose.More specifically, a “therapeutically effective amount” means an amountof active ingredients (e.g., a nucleic acid construct) effective toprevent, alleviate, or ameliorate symptoms of a disorder (e.g.,ischemia) or prolong the survival of the subject being treated.

It will be appreciated that compositions of the present invention may beattached to-, or included in medical devices, such as for promotingwound healing following implantation or promoting cell settling on theimplant.

Examples of medical devices which can be used in accordance with thepresent invention include, but are not limited to, intracorporeal orextracorporeal devices (e.g., catheters), temporary or permanentimplants, stents, vascular grafts, anastomotic devices, prostheticdevice, pacemaker, aneurysm repair devices, embolic devices, andimplantable devices (e.g., orthopedic (e.g., an artificial joint) andorthodental implants), aneurysm repair devices and the like. Otherdevices which can be used in accordance with the present invention aredescribed in U.S. Pat. Appl. No. 20050038498.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

For any preparation used in the methods of the invention, the dosage orthe therapeutically effective amount can be estimated initially from invitro and cell culture assays. For example, a dose can be formulated inanimal models to achieve a desired concentration or titer. Suchinformation can be used to more accurately determine useful doses inhumans.

Toxicity and therapeutic efficacy of the active ingredients describedherein can be determined by standard pharmaceutical procedures in vitro,in cell cultures or experimental animals. The data obtained from thesein vitro and cell culture assays and animal studies can be used informulating a range of dosage for use in human. The dosage may varydepending upon the dosage form employed and the route of administrationutilized. The exact formulation, route of administration, and dosage canbe chosen by the individual physician in view of the patient'scondition. (See, e.g., Fingl, E. et al. (1975), “The PharmacologicalBasis of Therapeutics,” Ch. 1, p. 1.)

Dosage amount and administration intervals may be adjusted individuallyto provide sufficient plasma or brain levels of the active ingredient toinduce or suppress the biological effect (i.e., minimally effectiveconcentration, MEC). The MEC will vary for each preparation, but can beestimated from in vitro data. Dosages necessary to achieve the MEC willdepend on individual characteristics and route of administration.Detection assays can be used to determine plasma concentrations.

Depending on the severity and responsiveness of the disorder to betreated, dosing can be of a single or a plurality of administrations,with course of treatment lasting from several days to several weeks, oruntil cure is effected or diminution of the disease state is achieved.

The amount of a composition to be administered will, of course, bedependent on the subject being treated, the severity of the affliction,the manner of administration, the judgment of the prescribing physician,etc.

Compositions of the present invention may, if desired, be presented in apack or dispenser device, such as an FDA-approved kit, which may containone or more unit dosage forms containing the active ingredient. The packmay, for example, comprise metal or plastic foil, such as a blisterpack. The pack or dispenser device may be accompanied by instructionsfor administration. The pack or dispenser device may also be accompaniedby a notice in a form prescribed by a governmental agency regulating themanufacture, use, or sale of pharmaceuticals, which notice is reflectiveof approval by the agency of the form of the compositions for human orveterinary administration. Such notice, for example, may includelabeling approved by the U.S. Food and Drug Administration forprescription drugs or of an approved product insert. Compositionscomprising a preparation of the invention formulated in apharmaceutically acceptable carrier may also be prepared, placed in anappropriate container, and labeled for treatment of an indicatedcondition, as further detailed above.

To improve therapeutic efficacy, compositions of the present invention(e.g., hydrogel) may be loaded with any pharmaceutical agent ofinterest. Release rate is pharmacokinetically controlled by the abovetailored formulations. Methods of loading hydrogels with pharmaceuticalsare well known in the art [see for example, drug inclusion in bovineserum albumin hydrogels described in Gayet and Fortier (1995) Art.Cells. Blood Subs. And Immob. Biotech. 23(5), 605-611].

Thus, for example, compositions of the present invention may enclosecomponents which are nonreactive to the composition (e.g., hydrogel).Examples of such nonreactive components may include drugs such asdisinfectants, chemotherapeutics, antimicrobial agents, antiviralagents, hemostatics, antiphlogistics, anesthetics, analgesics, ornutritional supplements; biopolymers such as peptides, plasma derivativeproteins, enzymes or mixtures thereof. In other words, componentsnonreactive to the hydrogel may be combined with the composition forhydrogel to provide stabilization or protection of these components.Such combined composition may be prepared, for example, by dissolving orsuspending the nonreactive components in the aqueous medium to be usedfor gelation before effecting the gelation.

Compositions of the present invention may, if desired, be presented in apack or dispenser device, such as an FDA-approved kit, which may containone or more unit dosage forms (e.g., 100 mg) such as for personalizeduse containing the active ingredient (e.g., precursor molecules whichare not yet cross-linked such as PEGylated denatured fibrinogen) andoptionally sterile disposable means for delivery (e.g., syringe) and forillumination (e.g., illuminator covers). The pack may, for example,comprise metal or plastic foil, such as a blister pack. The pack ordispenser device may be accompanied by instructions for administration.The pack or dispenser device may also be accompanied by a notice in aform prescribed by a governmental agency regulating the manufacture,use, or sale of pharmaceuticals, which notice is reflective of approvalby the agency of the form of the compositions for human or veterinaryadministration. Such notice, for example, may include labeling approvedby the U.S. Food and Drug Administration for prescription drugs or of anapproved product insert. Compositions comprising a preparation of theinvention formulated in a pharmaceutically acceptable carrier may alsobe prepared, placed in an appropriate container, and labeled fortreatment of an indicated condition, as further detailed above.

Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions illustrate the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Current Protocols in Immunology” Volumes I-IIIColigan J. E., ed. (1994); Stites et al. (eds), “Basic and ClinicalImmunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994);Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”, W.H. Freeman and Co., New York (1980); available immunoassays areextensively described in the patent and scientific literature, see, forexample, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578;3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533;3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521;“Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic AcidHybridization” Hames, B. D., and Higgins S. J., eds. (1985);“Transcription and Translation” Hames, B. D., and Higgins S. J., Eds.(1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “ImmobilizedCells and Enzymes” IRL Press, (1986); “A Practical Guide to MolecularCloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317,Academic Press; “PCR Protocols: A Guide To Methods And Applications”,Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategiesfor Protein Purification and Characterization—A Laboratory CourseManual” CSHL Press (1996); all of which are incorporated by reference asif fully set forth herein. Other general references are providedthroughout this document. The procedures therein are believed to be wellknown in the art and are provided for the convenience of the reader. Allthe information contained therein is incorporated herein by reference.

Example 1 Precursor Preparation

The blood-borne protein molecule fibrinogen is used to create a hybridprecursor molecule for tissue regeneration [Dikovsky et al.,Biomaterials 27(8), 14-96-506, 2006; Almany and Seliktar, Biomaterials,26(15), 2467-77, 2005]. Fibrinogen is the precursor to the commonlyknown blood clot protein, fibrin. The fibrinogen molecule contains a setof biological cell-signaling cues specific to cellular remodeling,including the cell-adhesive sequence Arg-Gly-Asp (RGD) and a proteasedegradation substrate [Herrick, Int J Biochem Cell Biol, 31(7)741-6,1999]. The fibrinogen molecules are transformed into abiosynthetic hybrid precursor using four sequential steps: 1) fibrinogendenaturation; 2) cleavage (optional); 3) PEGylation; 4) and 3-D matrixformation (optional). Each of these steps is detailed infra.

Step 1—Fibrinogen Denaturation: Denatured fibrinogen was prepared byincubating purified fibrinogen in a reducing a solution containing ahigh excess of Urea. A denatured fibrinogen is represented in FIG. 1 b.Briefly, purified bovine fibrinogen (Sigma-Aldrich, St. Louis, USA) wasdissolved in 8 M urea—phosphate buffered saline (PBS) at a concentrationof 3.5 mg/ml solution. Tris(2-carboxyethyl)phosphine hydrochloride(TCEP.HCl, Sigma, St. Louis, USA) was then added to the fibrinogensolution at a molar ratio 1.5:1 TCEP to fibrinogen cysteins. The 29-31cysteine residues present in the sequence of the fibrinogen protein weredenatured for 30 min in a stirred vessel at room temperature. Thedenatured fibrinogen fragments were visualized using SDS-PAGE andCoomassie®-blue staining (FIGS. 2 a-b).

Step 2—Fibrinogen Cleavage: Following Fibrinogen denaturation, thesolution was proteolytically cleaved using cyanogens bromine (CNBr).Basically, the denatured fibrinogen (Sigma, Steinheim, Germany) of step1 was dissolved in a 70% formic acid solution containing 17 mg/mlcyanogen Bromide (Aldrich) and incubated overnight in the dark at 25° C.The cleaved fibrinogen fragments were then dialyzed in 50 mM phosphatebuffered saline (PBS) at a pH=7.4 for 2 days at 4° C. with a twice-dailychange of buffer to remove all the CNBr and formic acid from thesolution. The dialyzed fragments were stored in PBS at 4° C. before thenext step of the precursor preparation procedure. The fibrinogenfragments were visualized using SDS-PAGE to confirm degradation products(FIG. 2 b).

Step 3—Denatured fibrinogen PEGyaltion: Cleaved Fibrinogen or uncleavedwhole fibrinogen was bound to monofunctional or multifunctionalpoly(ethylene glycol) (PEG) polymer using a Michael type additionreaction between the functional end groups on the PEG polymer and thereduced thiols on the denatured fibrinogen product. The PEGylationreaction scheme effected herein used linear PEG diacrylate(PEG-DA)(6-kDa or 10-kDa, Fluka Chemika GmbH, Buchs, Switzerland). Asolution of PEG-DA (280 mg/ml) in 50 mM PBS and 8M urea was added andreacted for 3 hours with cleaved or uncleaved denatured fibrinogen (ofstep 1 and/or 2). The molar ratio of PEG to fibrinogen cysteins is 5:1or more using linear PEG-DA, 6-kDa and 10-kDa. The PEGylated protein wasprecipitated by adding 4 volumes of acetone (Frutarom, Haifa, Israel).The precipitate was redissolved at 10 mg/ml protein concentration in PBScontaining 8 M urea and dialyzed against 50 mM PBS at 4° C. for 2 dayswith twice-daily changes of PBS (Spectrum, 12-14-kDa MW cutoff). Toestablish the total PEG-fibrinogen concentration, 0.5 ml of theprecursor solution were lyophilized overnight and weighed. The netfibrinogen concentration was determined using a standard BCA™ ProteinAssay (Pierce Biotechnology, Inc., Rockford, Ill.) and the relativeamounts of total PEGylated product (dry weight) to fibrinogen content(BCA™ result) were compared. A PEGylation efficiency scale was used todetermine percent PEGylation of the protein product. The scale assumes100% PEGylation when all 29 free thiols on the fibrinogen molecules (166kDa total) are bound to the functionalized PEGs. Schematic illustrationsof PEGylated fibrinogen are shown in FIGS. 3 a-b.

Step 4—3-D matrix formation: The polymer-modified denatured fibrinogenprotein (cleaved or whole) can be administered directly by injecting theprecursors locally or systemically, or alternatively, by creating a 3-Dmatrix for sustained local delivery of the fibrinogen degradationproducts to the surrounding tissue in a site-specific treatmentapplication. The use of a 3-D matrix for this purpose is done byimmobilizing the PEGylated fibrinogen to an existing biocompatiblematrix, or else by making a 3-D hydrogel matrix from the PEGylatedprecursors using polymerization of the additional reactive end groups onthe polymer.

The following provides an example using photoreactive chemistry tocreate denatured PEGylated fibrinogen hydrogels. PEG-modified denaturedfibrinogen (cleaved or whole) is formed into a solid polymer network bycross-linking between the free functional groups on the PEG-DA. Thesehydrogels are made from a precursor solution of denatured PEGylatedfibrinogen by a radical chain polymerization reaction of acrylate endgroups. The purified PEGylated precursor solution (2-3% PEGylatedpolymer—w/v) contains about 2 μM free acrylate groups for cross-linking.Additional PEG-DA (6-kDa or 10-kDa) can be added to increasecross-linking density of the PEGylated protein network as well as tominimize steric hindrance that may result in poor gelation (Gnanou etal, Macromolecules, 20, 1662-1671, 1987).

Briefly, the precursor solution was mixed with 1% (v/v) photoinitiatorstock olution made of 10% w/v Irgacure™ 2959 (Ciba Specialty Chemicals,Tarrytown, N.Y.) in 70% ethanol and deionized water. The solution wasplaced under a UV light (365 nm, 100 mW/cm²) for 5 min to polymerize.The molecular architecture and appearance of the hydrogels are shown inFIGS. 4 a-b.

Example 2 Denatured PEGylated Fibrinogen Degradation

The degradation of denatured or fragmented PEGylated fibrinogen intoPEGylated fibrinogen degradation products was verified experimentallyusing an in vitro degradation assay. Basically, PEGylateddenatured/fragmented fibrinogen fragments are formed into a matrix torecreate a 3-D matrix which can be implanted into a site-specificdefect.

Several variations of the PEGylated fibrinogen fragments are used inthese experiments, including cleaved and whole protein with either 6-kDaor 10-kDa PEG-DA attached to the protein. Protease degradation of thedenatured PEGylated fibrinogen is measured by colorimetrically assessingthe release of the PEGylated fibrinogen degradation products into thesupernatant during the course of the experiments. Cylindrical plugs arecast in 5 mm diameter silicon tubes from 100 μl aliquots ofPEG-fibrinogen precursor solution containing photoinitiator. Thedegradation is quantified by labeling the PEGylated fibrinogen witheosin-Y, which binds with high affinity to the fibrinogen, andquantifying the release of the fibrinogen resulting from enzymaticdissolution from the 3-D matrix. As the soluble fibrinogen degradationproducts are dissociated from the matrix, these released fragments arequantified by spectrophotometric measurements in an overlaying buffersolution.

Materials and Experimental Results

Casting of PEGylated fibrinogen—effected as described above.

Fibrinogen degradation—PEGylated fibrinogen 3-D samples were stained in5 mg/ml eosin-Y solution (Sigma-Aldrich, St. Louis, USA) for 2 days,washed, and transferred into 2 ml of either 0.01 mg/ml trypsin or 0.1mg/ml collagenase solution (Worthington, St. Louis, USA) containing 50mM PBS and 0.1% sodium azide. Absorbance values are measuredspectrophotometrically at 516 nm every 30 min for 3 hours. After thelast time point, each hydrogel was hydrolytically dissociated, andabsorbance values were recorded as 100% degradation.

Results

FIGS. 5 a-d depict the results from degradation experimentsdemonstrating that the percent degradation of PEG-fibrinogen precursors(i.e., ratio of release PEG-fibrinogen to total PEG-fibrinogen) wasaffected by the molecular weight of the grafted PEG, the composition ofPEG to fibrinogen, and the molecular configuration of the denaturedfibrinogen (whole or cleaved). The degradation of the PEG-fibrinogenprecursors in collagenase (0.1 mg/ml, FIGS. 5 c-d) or trypsin (0.01mg/ml, FIGS. 5 a-b) is shown.

The kinetics data reveals that precursors made with 10-kDa PEG, arereleased faster from the matrix than precursors made with 6-kDa PEG.Addition of PEG-DA to the precursor solution resulted in significantlyslower degradation kinetics (n=5, p<0.01). Changing the PEG molecularweight from 6-kDa to 20-kDa resulted in accelerated degradation in thepresence of 0.05 mg/ml trypsin (n=6, p<0.05, data not shown). ThePEGylated precursor responsiveness to differing enzyme concentrationswas assessed by measuring degradation in serial dilutions of collagenaseor trypsin following 30 minutes of incubation (not shown). The resultsfrom these experiments confirm dose-dependent relationship between ofthe degradation kinetics of the PEGylated precursors and the enzymeconcentrations. The results were also verified by degrading PEGylatedfibrinogen fragments and analyzing them using SDS-PAGE (data not shown)which revealed smaller fragments of PEGylated degradation productscompared to the non-degraded PEGylated precursors.

Example 3 Tissue Culture Assay

The protease-mediated release of the denatured PEGylated fibrinogenproducts from intact 3-D materials may have a profound chemotacticeffect on in vitro tissue cultures of various types. It is assumed thatwhen released from the 3-D matrix, the denatured PEGylated fibrinogendegradation products may facilitate the chemotactic invasion of cells,including vascular smooth muscle cells, chondrocytes, and embryonic stemcells, into the 3-D matrix containing the bound PEGylated fibrinogen.Therefore, the chemotactic potential of the denatured PEGylatedfibrinogen degradation products was assayed by an in vitro tissueculture assay designed in part for this task.

Chemotaxis assay—A cell invasion assay was employed using smooth muscletissue encapsulated by a PEGylated fibrinogen matrix (10 kDa PEG). Densetissue constructs composed of smooth muscle cell-seeded collagen gelsplaced inside PEG-fibrinogen hydrogels. The smooth muscle tissueconstructs were made from a solution of 5×DMEM, 10% FBS, reconstitutedtype-I collagen solution in 0.02 N acetic acid (2 mg/ml), and 0.1 M NaOHwith dispersed smooth muscle cells (3×10⁶ cells/ml, cell were isolatedfrom bovine aortic tissue explants according to standard protocols[Seliktar et al, Ann Biomed Eng, 28(4), 351-62, 2000]. The collagen gelswere placed in 300 μl of PEG-fibrinogen precursor solution (withphotoinitator) in a 48-well plate and exposed to UV light for 5 minutes.The polymerized hydrogels encapsulated the tissue during the gelationprossess. The encapsulated tissue constructs were supplemented with 600microliters of culture medium containing DMEM (Gibco), Pen-strep, andfetal bovine serum (FBS, Gibco). The constructs were placed in acontrolled temperature and CO₂ incubator and medium was replenishedevery other day. The cells were imaged daily using a phase contrastmicroscope to determine the level of cell migration from the edge of thetissue and into the PEG-fibrinogen hydrogel. Histological staining forcell morphology [Hematoxylin and Eosin (H&E)] were performed to assessthe morphology of invading cells.

Cartilage explant experiments were performed using similar methods aswith the smooth muscle tissue. In this case 1-mm slices of articularcartilage explants, were isolated from sheep knee joints and immersed inthe precursor solution prior to being polymerized with UV light (365-nm,700 μW/cm²). The encapsulated explants were cultivated with twice-weeklyreplenishment of medium and periodic imaging as detailed above.Histological staining for proteoglycans (Safranin-O), cell morphology(H&E), and immuno-staining for type I and type II collagen wereperformed to assess the phenotype of invading chondrocytes and theexplanted specimen.

Results

FIGS. 6 a-j show cellular invasion from vascular tissue intoPEG-Fibrinogen containing hydrogel matrix. Initially, the smooth musclecells were contained within the collagen gel construct (FIG. 6 a).Several hours following casting, the cells began to proteolyticallydegrade the matrix and release PEGylated fibrinogen degradationproducts. The chemotactic effect of the peptides caused a massivecellular invasion into the dense PEG-fibrinogen matrix surrounding thetissue mass (FIG. 6 b), which lasted for the duration of the cultureperiod as more PEGylated fibrinogen was proteolytically degraded by thecell-secreted enzymes (FIG. 6 c). Of note, during the first 96 hours ofculture, the rate of cellular degradation and invasion was nearlyconstant and proportional to the molecular weight of the PEG-fibrinogenprecursors (data not shown). Following this period, the degradation andinvasion into the matrix was more aggressive as indicated by a sharpincrease in the rate of cellular invasion after one week (91% increase,n=5, p<0.01, FIG. 7 a). Overall, the rate of cellular degradation andinvasion into 10-kDa PEG-fibrinogen hydrogels was significantly higherthan the rate of degradation and invasion into 6-kDa hydrogels (n=4,p<0.05) (FIG. 7 b). The additional PEG-DA used to polymerize thehydrogel network significantly altered the rate of cellular degradationand invasion into the denatured PEGylated fibrinogen containing matrix(FIGS. 6 a-j, FIG. 7 a-b).

Other tissue types demonstrated similar responses when encapsulated inthe above degradable matrix. Studies using both embryonic stem cells andcartilage tissue explants exemplified the chemotactic response of bothtissue types to the PEGylated degradation products (FIGS. 7 c-d,respectively).

Example 4 In Vivo Osteogenesis Mediated by PEGylated FibrinogenDegradation Products

The osteoinductive properties of denatured PEGylated fibrinogendegradation products in osseous regeneration were studied in asite-specific bone defect.

Materials and Experimental Procedures

Implant Fabrication: Acellular cylindrical plugs were cast in 3-mmdiameter silicon tubes using 88 μl aliquots of PEG-fibrinogen precursorby a photopolymerization reaction of acrylate end groups. AdditionalPEG-DA (3% or 5% w/v) was added to the precursor solution in order toreduce the susceptibility of the PEGylated fibrinogen backbone toproteolytic degradation (Table 1, below). The final ratio of PEG tofibrinogen monomer was 25:1, 100:1, and 150:1 for the 0% PEG-DA, 3%PEG-DA, and 5% PEG-DA, respectively. These different compositionscorrespond to fast, intermediate and slow degrading hydrogels(respectively) as indicated in Table 1 below. The precursor solution wasmixed with 0.1% (v/v) photoinitiator stock solution consisting of 10%w/v Irgacure™ 2959 (generously donated by Ciba Specialty Chemicals,Tarrytown, N.Y.) in 70% ethanol and deionized water. The solution wasplaced under a UV light (365 nm, 40-50 mW/cm²) for 5 min to polymerize.The pre-cast hydrogels were stored in 50 mM PBS containing 2%penicillin-streptomycin (Biological Industries, Israel) for 5 hrs priorto implantation.

Rat implantation—Approval of the Institutional Review Board of theRappaport Faculty of Medicine of the Technion, Israel Institute ofTechnology was obtained prior to initiation of the performed experimentsand all experiments were performed in accordance with the guidelines setout by the Technion animal care committee. Female Sprague-Dawley rats(32 animals altogether) aged 3 to 4 months and weighting about 250 gm)were adapted to cage life for 5 days prior to the surgery. The weight ofthe animal was monitored during this period to ensure stability andproper adaptation. The animals were fed regular lab chow and had accessto tap water ad libitum. They were anesthetized with a combination ofKetamine 120 mg/kg and Xylazine 17 mg/kg. During the surgical procedurethe animal was placed on a warm plate to maintain body temperature (andprevent hypothermia). The right tibia was shaved and wiped with polydinetincture solution. The mid-portion of the right tibia was exposed fromthe anterior medial side by longitudinal incision. An external fixationdevice was placed proximal and distal to the mid-section of the tibiaaccording to published protocols [Srouji et al, Cell Tissue Bank, 5(4),223-30, 2004]. Briefly, two needles were drilled into the proximal (21G)and distal (23G) segments of the tibia and connected to two externalfixation apparatuses so as to stabilize the bone (FIGS. 8 a-d). A 7-mmgap was excised using a high-speed disk saw in the portion between theproximal and distal needles of the fixation devices. The ipsilateralfibula was left intact. A PEG-fibrinogen plug (3-mm diameter and 7-mmlong) was inserted into the site of the defect and the surroundingperiosteum as well as the subjacent fibrous tissue were wrapped aroundand sutured to secure the plug in place (FIG. 8 d). The incisional woundwas sutured with a nylon surgical thread. The rats were givenprophylactic antibiotics (ampcillin 0.1 gr/100 g). They were x-rayedshortly after the surgery and thereafter were evaluated at weeklyintervals by x-ray screening. They were housed in spacious cages so asto allow relative free ambulation during the entire postoperativefollow-up period. At the end of the 5 week evaluation period, the ratswere sacrificed with CO₂ and the right tibial bones were harvested forhistological evaluation.

Results

Hydrogel comprising PEGylated fibrinogen with various degrees ofproteolytic resistance based on poly(ethylene glycol) and fibrinogencomposition were designed for slow, intermediate, and fast releasekinetics of the degradation products. The hydrogels were implanted intosite-specific defects of rats' tibiae without additional osteoinductivefactors with the rational that the matrix will displace the normalfibrin clot while sustaining a similar healing effect in the defect sitefor a longer duration by releasing the denatured PEGylated fibrinogendegradation products. Histological and x-ray results described indetails hereineblow have confirmed that following 5 weeks ofimplantation, the extent and distribution of newly formed bone in thedefect strongly correlates with the erosion pattern of the implantedmaterial and subsequent release of degraded PEGylated fibrinogen to thelocal tissue environment. When compared to nonunions in untreatedcontrol animals, the rats implanted with the intermediate-degradingPEG-fibrinogen materials displayed osteoneogenesis. These data supportthe suggestion that the release of denatured PEGylated fibrinogendegradation products provides inductive properties. In thissite-specific bone defect model, the sustained release of denaturedPEGylated fibrinogen degradation products facilitates the osteogenicresponse in the injury site. TABLE 1 Summary of Treatment CohortsDegradation Degradation Rate Rate in 0.01 mg/ml In 0.1 mg/ml CompositionTrypsin Collagenase Degra- PEG:Fibrinogen (% Wt (% Wt Group dation ratioLoss/min^(1/2)) Loss/min^(1/2)) Control N/A Empty gap N/A N/A 1 Fast 25:1 6.960 7.878 2 Inter- 100:1 1.168 1.928 mediate 3 Slow 150:1 0.2920.351

Specific analyses effected to substantiate the osteoindcutive effect ofthe compositions of the present invention

Histological Analysis: Following a final radiographic evaluation, theright tibia of each rat was carefully excised in its entirety. Thesamples were fixed in buffered, neutral 10% formalin solution for 10days and then decalcified in 10% formic acid for 10 days. The specimenswere trimmed so as to include the implant site and the adjacent bonetissue on either side of the defect. After rinsing in PBS, the specimenswere dehydrated in increasing concentrations of ethanol in deionizedwater (70% to 100%). Specimens were embedded in extra-large paraffinblocks, which were sectioned at 6 μm, fixed on poly-1-lysine coatedglass slides, and stained with hematoxylin and eosin (H&E).

Resulting Osteogenesis: Newly formed bone in the site-specific defectsof the tibiae was radiographically observed as early as three weekspostoperatively. When compared to control rats, large amounts of newbone were apparent in the defects of the treatment-2 animals by 5 weeks(data not shown). This contrasted with the lack of radiographicallydetectable bone in the defects of the treatment-1 (fast degrading),treatment-3 (slow degrading) and control rats. The histologicalexamination confirmed that the rats treated with theintermediate-degrading treatment 2 exhibited the most extensive andwidespread osteoneogenesis in and nearby the defect site. Thelongitudinally, H&E-stained sections of the tibiae revealed that theextent of regenerated bone in the site-specific defects ranged frompartial to total bridging of the gap (FIGS. 9 a-c). Osteoneogenesis wasobserved at both the endosteal and subperiosteal aspects. When observedunder polarized light, the birefringent pattern of the preexistinglamellar-fibered cortical bone sharply contrasted to that of thewoven-fibered boney trabeculae, characteristic of newly depositedosseous tissue. The newly formed subperiosteal bone at the osteotomysites was contiguous with the boney trabeculae, which were for the mostpart rimmed by cuboidal osteoblasts on their inner front. Theendosteally formed bone was as well continuous with newly formedtrabeculae, which extended into the defect site. Randomly scatteredadipocytic islands were present in between the trabeculae of the newlyformed woven-fibered bone. In but a few samples, the medullary cavitycontained some fibrous tissue proximal to the osteotomy site.

In those cases in which there was total osseous bridging of thesite-specific defect, the implant had been entirely resorbed andreplaced by lamellar-fibered bone with an atypical pattern of Haversian,essentially, osteonal bone. The newly formed bone was uninterrupted fromone end to the opposite end of the osteotomies (FIG. 9 c), consisting ofbirefringent, lamellar-fibered, compacta-type bone with a moderatenumber of vessels within the Haversian system (data not shown),characteristic of mature bone. In those instances in which there was buta partial bridging of the defect, there was often endochondralossification of cartilaginous islets. To exemplify, FIG. 10 aillustrates a typical endochondral cap at the medial aspect of theregeneration front: Hypertrophic chondrocytes were focally present inthe cartilaginous cap, which was enclosed by a thin, perichondrium-likefibrous tissue with parallel-oriented mature fibrocytes at its leadingedge (FIGS. 10 a-b).

Mechanism of Neoosteogenesis—As shown above only the intermediatedegrading PEG-fibrinogen hydrogel treatment (treatment-2) causedextensive new bone formation at the site of the boney defect.Nevertheless, judging from just the histological findings it is clearthat the PEGylated fibrinogen material is endowed with osteoinductiveproperties and that the macrophages that erode the denatured PEGylatedfibrinogen containing matrix slowly release osteoinductive denaturedPEGylated fibrinogen degradation products to act as an eroding front forosteoneogenesis to occur in as much as the gels slowly give way for thenewly generating bone. There is consistency with the observations thatfaster degrading hydrogels do not provide synchronized release with thenatural healing rate in a rat site-specific bone defect, which typicallyrequires 4 to 5 weeks to heal completely. It is important to note thatin this study, the gel composition was deliberately chosen to coincidewith the optimal degradation rate and healing kinetics of this type ofinjury. Even though slow and fast degrading gels are suboptimal for thistype of injury, it is the ability to regulate the degradation andrelease of the PEGylated fibrinogen degradation products that allows thepresent this technology to treat any number of different type of injuryin humans.

It was observed that the fragments released from the PEG-fibrinogenhydrogel facilitate a prolonged osteogenic response within thesite-specific defect. In particular, a sustained release of thedenatured PEGylated fibrinogen degradation products explain the extentof the oesteogenic response in the treatment of group 2 rats. There isample evidence that fibrinogen and fibrin degradation products arepotent agonist of wound healing [Thompson et al, J Pathol, 165(4),311-8, 1991; Rybarczyk et al, Blood, 102(12), 4035-43, 2003], especiallyas concerns endothelial cells [Lorenzet et al, Thromb Haemost, 68(3),357-53, 1992; Bootle-Wilbraham et al, Angiogenesis, 4(4), 269-75, 2001]and fibroblasts [Gray et al, Arm J Respir Cell Mol Biol, 12(6), 684-90,1995; Gray et al, J cell Sci, 104(Pt2), 409-13, 1993]. In fact, fibrinhas been evidenced to induce an osteogenic response in bone defectsfilled with osteoconductive materials [Le Guehennec et al, J Mater SciMater Med, 16(1), 29-35, 2005; Abiraman et al, Biomaterials, 23(14),3023-31, 2002; Kania et al, J Biomed Mater Res, 43(1), 38-45, 1998].Moreover, if fibrinogen fragments do not possess osteoinductivequalities, we would expect a similar outcome to that demonstrated byother researchers who utilize the inert biomimetic ingrowth matriceswithout added growth factors. Pratt et al. have reported that erodingfibrin-mimetic hydrogels are unable to support new bone formation whenoccupying a size-specific calvarial defect in the absence of theosteoinductive BMP-2 [Pratt et al, Biotechnol Bioeng, 86(1), 27-36,2004]. Likewise, Lutolf et al. have demonstrated similar resultsemploying a collagen-mimetic biosynthetic ingrowth matrix withoutosteoinductive BMP-2 [Lutolf et al, Nat Biotechnol, 21(5), 513-8, 2003].In comparison, the extent of osteoneogenesis using PEG-fibrinogencontaining hydrogels without added osteoinductive growth factors canonly be explained by an osteoinductive role of the PEGylated fibrinogendegradation products in as much as it is unlikely that the PEGconstituent has osteoinductive qualities.

The unprecedented osteoneogenesis observed in this study is attributedto the released fragments of PEGylated fibrinogen degradation productsand not necessarily to the intact matrix. A sustained presentation ofmildly osteogenic PEGylated fibrinogen fragments could account for theprolonged osteogenic response over the 5 weeks of the healing period.This explanation is consistent with the fact that most of theosteoneogenesis in the treatment-2 defects occurs at least severalhundred microns from the eroding surface of the hydrogels (FIG. 11 a),the latter being consistently surrounded by an inflammatory infiltrate.Even the slow degrading hydrogels (treamtnet-3) induce some mildosteogenic response around the implant, presumably because of thereleased fragments of PEGylated fibrinogen (data not shown). From thispoint of view, there is no evidence of osteoneogenesis in the fastdegrading hydrogel-treated animals (treatment-1), suggesting that rapiddissolution of the PEGylated fibrinogen fragments does not enableadequate new bone formation.

Example 5 In Vivo Chondrogenesis Mediated by PEGylated FibrinogenDegradation Products

The osteoinductive properties of denatured PEGylated fibrinogendegradation products in cartilage regeneration were studies in acritical size osteochondral defect.

Materials and Experimental Procedures

Precursor Fabrication—PEG-fibrinogen precursor was made from PEGylatedfibrinogen according to the method detailed above. The implant solutionwas modified with additional PEG-DA in order to reduce thesusceptibility of the PEGylated fibrinogen backbone to proteolyticdegradation. The final ratio of PEG to fibrinogen monomer used was150:1, 200:1, and 350:1. The aliquots of PEG-fibrinogen precursor weremixed with 0.1% (v/v) photoinitiator stock solution including 10% w/vIrgacure™ 2959 (generously donated by Ciba Specialty Chemicals,Tarrytown, N.Y.) in 70% ethanol and deionized water. The polymerizationof the solution was tested under UV light (365 nm, 40-50 mW/cm²) for a 5min duration to determine if the solution polymerizes prior toimplantation. Once polymerization was verified, the precursor solutionwas ready for in situ polymerization. In the osteochondral defect model,the implant is polymerized directly in the 6-mm defect using a UV sourceand light guide (FIGS. 12 a-d).

Sheep Implantation: Adult, skeletally mature sheep (weighing on average70 kg) were adjusted to cage life one week prior to operation. Followinga 24-h fast and pre-medication by i.v. infusion of ketamine-Hcl 10 mg/kgand xylazine 0.05 mg/kg, induction by i.v. administration of propofol4-6 mg/kg, the animal was intubated. After intubation, includingmaintenance by inhalation of isoflurane 1.5-2% and ventilated bypositive pressure of 100% O₂ by volume control, a bolus of 0.1 mgfentanyl was administered to the animal immediately prior to surgery.Postoperative analgesis by p.o. tolfine 2-4 mg/kg X3/d was conducted.The animals received 2.5 g metamizol and 1 g cefazoline twice dailyuntil the third postoperative day. The experiments were performed on theright stifle joint of the hind leg of the sheep. The leg was sterilelydraped and opened by a parapatellar anterolateral approach. The paterllawas dislocate medially, and the femoral condyle was exposed. Using a6-mm custom made punch and drill tool, two defects, 1 and 2.5 cm distalfrom the intercondylar notch, were introduced in the weight bearing zoneof the femoral condyles (FIG. 12 a). The defects were created with thepunch and both the subchondral bone and cartilage were completelyremoved with the drill bit. There was some intraoperative bleeding fromthe subchondral bone which was subdued using a sterile gauze. Into thenon-bleeding defect sites the PEG-fibrinogen solution was sterilelyinjected and polymerized in situ using a hand-held, UV light source(FIG. 12 d). Wound closure was then performed in layers. An externalplaster (Scotch/Soft Cast, 3M HealthCare, St. Paul, Minn., USA) wasapplied on the stifle and ankle joint for 5 days. The animals' cageactivity was limited in order to reduce joint loading. After the removalof the plaster, the sheep was allowed to move freely, and given abalanced diet twice a day. At the end of the 4-month evaluation period,the animals were sacrificed and the stifle joints of the hind leg washarvested for gross observation, histology, and immunohistochemistry.Following sacrifice, the distal femur was removed and placed in 10%neutral buffered formalin. After 24 h, the areas of condyles containingthe defects were dissected and placed back in 10% formalin for 4 days.The decalcified specimens were embedded in paraffin and sectioned to4-mm-thick slices.

Results

Osteochondral defects treated with PEGylated fibrinogen hydrogelsexhibited regeneration of articular cartilage as observed by grossobservation after 4 months (FIGS. 13 a-d). FIG. 13 a-b show the fourdefects on the day of operation, in the patellar notch (FIG. 13 a) andin the condyle (FIG. 13 b). After four months, the same defects areshown in the patellar notch (FIG. 13 c) and the condyle (FIG. 13 d).Only the medial patellar notch defect (FIG. 13 c, right) did not exhibitnew cartilage formation. All the other defects, which were treated, hadnew cartilage growing on the joint surface.

Histological evaluation of these defects revealed extensive newcartilage formation in the defect region, alongside new bone formationaround the eroding implant (FIGS. 14 b-d). Empty defects exhibitedfibrocartilage and excessive scar tissue formation (FIG. 14 a). Theextent of released PEGylated degradation products and subsequent erosionof the hydrogel implant was dependent on the ratio of PEG to fibrinogen;hydrogels with more PEG exhibited slower erosion and slower release ofthe therapeutic PEGylated fibrinogen degradation products. FIGS. 14 a-dsummarizes the relationship between the molar ratio of the PEG tofibrinogen and the erosion patterns of the hydrogel implant. Furtherhistological staining of the sections using stains for type I collagen(FIG. 15 a-d), proteoglycans (FIG. 16 a-d), and type II collagen (FIG.17 a-d) showed characteristic staining of articular (hyaline) cartilageabove the newly formed boney bridge overtop the eroding implant. Type Icollagen staining was performed (FIG. 15 a-d) to verify new boneformation in the site of the defect, around the eroding implant.Proteoglycan staining was performed (FIG. 16 a-d) to verify generationof hyaline cartilage in the treated defects (FIG. 16 b-d) whereascontrol treated defects were not positive for proteoglycans (FIG. 16 a).Type II collagen staining was performed (FIG. 17 a-d) to confirm thatthe composition of the cartilage surface is consistent with thecomposition of hyaline cartilage; in those treated samples that werestained, the newly formed cartilage surface was found to be positive fortype II collagen (FIG. 17 b-d).

Osteogenesis and Chondrogenesis: Newly formed cartilage and bone in thecritical size defect was apparent in all three treatment conditionsafter 3-4 months (FIGS. 14 b-d). On the other hand, control defects werefilled with scar tissue and did not show any signs of chondrogenesis(FIG. 14 a). It is suggested that the slow-released implants may be moreeffective in healing the injury based on the quality of the articularcartilage formed in the treated defect (FIGS. 19 a-d). Similar with thebone study, it is clear that the PEGylated fibrinogen material isendowed with both inductive properties for bone and cartilage repair andthat the macrophages that erode the denatured PEGylated fibrinogencontaining matrix slowly release osteoinductive denatured PEGylatedfibrinogen degradation products to act as an eroding front forosteoneogenesis and chondrogenesis to occur in as much as the gelsslowly give way for the newly generating bone and cartilage. Here again,it is important to note that we arbitrarily choose the composition ofgel to coincide with the optimal degradation rate and healing kineticsin this type of injury. Consequently, the healing kinetics of thisinjury are such that it was possible to observe cartilage regenerationin all the three treatment conditions whereas the control group (emptydefect) was clearly not capable of creating new cartilage. Even thoughthese compositions were optimal for this type of injury in sheep, it isthe ability to regulate the degradation and release of the PEGylatedfibrinogen degradation products that affords this technology with theversatility for treating the same injury in humans.

Fragments released from the PEG-fibrinogen hydrogel facilitated aprolonged osteogenic and chondrogenic response within the subchondrollesion. As with the rat study above, a sustained release of thedenatured PEGylated fibrinogen degradation products explain the extentof the oesteogenic and chondrogenic response in the treated animals. Theobserved chondrogenesis in particular is attributed to the releasedfragments of PEGylated fibrinogen degradation products and not to theintact matrix. A sustained presentation of mildly inductive PEGylatedfibrinogen fragments could account for the prolonged chondrogenicresponse over the 4 months of the healing. This explanation isconsistent with the fact that most of the regeneration of bone andcartilage in the treated defects occurs at least several hundred micronsfrom the eroding surface of the hydrogels (FIGS. 18 a-b), the latterbeing consistently surrounded by an inflammatory infiltrate.

It is particularly impressive to see the extent of the seamlessintegration between the margins of the defect and the newly formedcartilage in the treated animals (FIGS. 20 a-f). In some cases, it wasdifficult to gauge where the original margins of the defect ended andthe newly formed tissue began. Similarly, the newly formed bone was verywell integrated with the surrounding existing bone tissue of the injurymargins. This is an important indicator of the quality of the healingthat is achievable by means of the PEGylated denatured fibrinogendegradation technology.

Conclusions: Injecting implants that erode and release PEGylateddenatured fibrinogen degradation products into an osteochondral defectsite can promote the repair of the articular cartilage surface throughinduction and synchronized release of the inductive fibrinogenfragments.

Example 6 Controlling Three-Dimensional Neurite Outgrowth UsingPEG-Fibrinogen Hydrogels

To test the potential of the PEG-fibrinogen scaffold material tofacilitate nerve regeneration, a chicken embryo dorsal root ganglion(DRG) outgrowth model was used. In the initial stage of nerveregeneration fibrin provides Schwann cells environmental cues forproliferation, thus ensuring that there are enough cells to associatewith the regenerating neurons. In the absence of fibrin, Schwann cellscan then differentiate and re-myelinate the newly formed axons.Accordingly, this model implies that the untimely persistence of fibrinin the injury site can interfere with the delicate timing of the nerveregeneration process and disrupt the construction of functional nervetissue. Therefore the ability to control the degradation and removal ofthe fibrin matrix is crucial for enabling successful nerve regeneration.

The synthetic PEG component provides the desired physical properties andcontrollable degradation characteristics. The natural fibrinogencomponent of the biosynthetic matrix supplies cues that regulate Schwanncell proliferation and migration and therefore will likely influencere-myelination of the regenerated axons. An additional advantage of thePEGylated fibrinogen approach is that it enables the control of therelative bioactivity of the fibrinogen degradation products based on therationale that covalently bound PEG can decrease the accessibility toactive sites on both intact and degraded fibrinogen molecule. Hence, aPEGylation strategy offers control over fibrinogen degradation,bioactivity, and molecular architecture of the nerve guidance conduit(NGC) cell ingrowth matrix.

In addition, the present inventor has shown that it is possible tocontrol the biodegradation of the fibrinogen matrix by changing relativeamounts of fibrinogen and PEG in such a system. To this end, Dikovsky etal. showed that increased PEG-DA concentrations in the PEGylatedfibrinogen hydrogel decreased proteolytic susceptibility of the proteinbackbone and thus delayed the PEGylated fibrinogen biodegradation[Dikovsky D. et al. Biomaterials 2006;27(8):1496-506]. The PEGylatedfibrinogen system also presents additional advantages for nerveregeneration in that therapeutic growth factors can easily beencapsulated and enmeshed in the dense polymeric network of the hydrogelduring the polymerization process. The encapsulation of factors fornerve regeneration could provide neuron-specific signals beyond theinherent biological and structural provisions of the PEGylatedfibrinogen hydrogel network. One of the most vital neurotrophins inneuronal development and regeneration is nerve growth factor (NGF).Schwann cells produce NGF, a 26 kDa dimmer, in their immature phaseduring early development and after post-injury dedifferentiation inmature nerves. Accordingly, it is important that NGF be an integral partof the nerve guidance implant material.

Materials and Experimental Methods

Dorsal Root Ganglia Experiments: DRGs were dissected from E9-E11 chickenembryos and collected in PBS with 1% penicillin-streptomycin (Biologicalindustries, Kibbutz Beit Haemek, Israel). Fibroblast contamination ofthe DRGs was minimized by pre-plating the DRGs for one hour in MEM withGlutamax I medium (Gibco, Grand Island, N.Y., USA) containing 1%penicillin-streptomycin and 10% fetal calf serum (FCS) (Biologicalindustries, Kibbutz Beit Haemek, Israel). The pre-plate DRGs were thenphysically removed from the culture dish and entrapped in hydrogelconstructs prepared from a precursor solution of PEGylated fibrinogen(prepared as described in Example 1) and photoinitiator. Briefly, theprecursor solution was mixed with 1% (v/v) photoinitiator stock solutionmade of 10% (w/v) Irgacure™ 2959 (Ciba Specialty Chemicals, Tarrytwon,N.Y.) in 70% ethanol and deionized water. The solution was thencentrifuged at 14,000 RPM for 1 minute before being used to entrap theisolated DRGs. The entrapment procedure involved gently placing theintact DRGs into a 48-well plate containing the precursor solution. The48-well plate was first pre-coated with 100 μl polymerized PEGylatedfibrinogen in order to prevent cell growth on the bottom of the well.Each DRG was placed into 200 μl PEGylated fibrinogen solution andpolymerized under a UV light (365 nm, 4-5 mW/cm²) for 5 minutes. Afterhydrogel polymerization, the entrapped DRGs were visually inspected toensure 3-D encapsulation in the biosynthetic matrix (FIGS. 25 a-c).Culture medium was immediately added to the polymerized hydrogels (500μl in each well) and changed every two days. The culture medium wascomprised of MEM with Glutamax I medium containing 1%penicillin-streptomycin and 10% FCS. Unless otherwise indicated, themedium was supplemented with 50 ng/ml 2.5S mouse nerve growth factor(mNGF) (Alomone labs LTD., Jerusalem, Israel).

Quantitative Outgrowth Measurements: Cellular outgrowth from the DRGinto the transparent PEGylated fibrinogen hydrogel was recorded duringthe four-day duration of the experiment. Each DRG construct wasdocumented with digital images taken daily using a Nikon TE2000 phasecontrast microscope with a 4× objective and a digital CCD camera(Jenoptik, Germany). Quantitative neurite outgrowth measurements wereobtained directly from the digital phase contrast micrographs usingImageJ software. Neurites, which can be identified by theircharacteristic sprouting morphology, were measured from the base (outermargin of the DRG) along their length and up to the tip. Up to a totalof 80 measurements were made for each DRG construct, according to theability to trace continuous neurites. The mean DRG neurite outgrowth wasthen calculated for each individual DRG construct by averaging the 80measurements of each construct (n=1). The average neurite outgrowth foreach treatment was calculated using the mean DRG neurite outgrowth data.

Histology and Immunofluorescence: Preparation of the DRG specimens forhistological and immunofluorescence evaluation involved fixation in 4%paraformaldehyde (Gadot, Haifa, Israel) for 20-30 min, PBS rinses, andovernight cryoprotection in a 30% sucrose solution (in PBS) at 4° C.Each fixed construct was then slow-frozen in Tissue-Tek® O.C.T Compound(Sakura Finetek, Torrance, Calif., USA) using liquid nitrogen cooledisopropanol (Gadot, Haifa, Israel). Frozen constructs were stored in adeep freezer (−80° C.) for up to three months. The specimens weresectioned orthogonally into 30-μm thick slices using a cryostat andmounted on Polysin™ slides (Menzel-Glaser, Braunschweig, Germany). Priorto staining, the slides were air dried at RT for 2 hours and stored at−20° C. Hematoxylin and Eosin (H&E) staining (Sigma, St. Louis, Mo.,USA) was performed according to standard manufacturer's protocols.

Immunofluoerscence labeling of the 30-μm thick specimens involvedtreatment with 0.3% Triton® X-100 (Bio Lab LTD., Jerusalem, Israel) for5 min at RT and incubation in blocking solution containing PBS and 1%glycine, 10% horse donor serum (HDS) (Biological industries, KibbutzBeit Haemek, Israel) and 0.1% Triton® X-100 for 30 min at RT. Thesections were double stained with primary antibodies againstβPIII-tubulin, (G712A, Promega, Madison, Wis., USA) and s100 (S2644,Sigma, St. Louis, Mo., USA). The primary antibodies were diluted inblocking solution (1:1000 dilution for βIII-tubulin and 1:200 dilutionfor s100) and incubated overnight at 4° C. in a humidity chamber. Thesections were rinsed and incubated for 30 minutes at RT withfluorescently conjugated secondary antibodies, including 1:250 dilutedgoat anti-mouse Cy3 (Chemicon International, Temecula, Calif., USA) forβIII-tubulin and 1:300 diluted goat anti-rabbit FITC (JacksonImmunoresearch Laboratories INC., west Grove, Pa., USA) for s100. Anuclear counter-stain was incorporated directly into the secondaryantibody staining solution using a 1:500 diluted DAPI stock solution(Sigma Aldrich, St. Louis, Mo., USA). Following incubation, sectionswere rinsed with PBS and mounted with FluoromountG (SouthernBiotechnology Associates, INC., Birmingham, Ala., USA).

Statistical analysis: Statistical analysis was preformed on data setsfrom at least two independent experiments. Depending on the data set,treatments were compared by single-factor ANOVA, two-factor ANOVA, orpaired student t-test. Statistical significance was accepted for p<0.01.

Experimental Results

DRG Outgrowth: Tissue constructs were prepared by entrapping DRGs insidePEGylated fibrinogen hydrogels (FIGS. 21 a-c) and cultivating them forup to one month in a CO₂ incubator. Cellular outgrowth from the DRG wasvisible in phase contrast micrographs and histological H&E sections(FIGS. 22 a-d). Throughout the experiment, cells from the DRG invadedthe PEGylated fibrinogen hydrogel and eventually occupied the entire gel(not shown). Phase contrast micrographs show the distinct spatialorganization and orientation of the invading DRG cells into thePEGylated fibrinogen matrix after two days (FIG. 22 a). A highmagnification of this organization is shown in FIG. 22 b, where longthin processes (neurites) extending out of the DRG are accompanied bynon-neuronal cells (dark circular spots) that emerge from the DRG coreand align along the neurite extensions. The non-neuronal outgrowth fromDRGs (FIG. 22 b, arrowhead) was shown to lag after neurite extensions(FIG. 22 b, arrow). Histological cross-sections (30 μm) of the DRGconstructs following four days of culture stained with H&E showedsimilar cellular invasion characteristics (FIGS. 22 c-d).

The arrangement of non-neuronal (glial) cells invading from the DRG andaligning with the neurites resembled the in vivo spatial organization ofneurons and their associated Schwann cells. In order to identify thedifferent invading DRG cells in these experiments, neurites and Schwanncells were both labeled with neuronal and glial immunofluorescentmarkers. Immuno-detection in 30 μm thick cross-sections of the DRGconstructs was preformed with the neuronal marker βIII-tubulin antibodyand the Schwann cell marker s100 antibody. The labeling clearly showsextending neurites originating from the DRG into the matrix, andassociated Schwann cells in close proximity to the invading neuronalcells (FIGS. 23 a-c). Higher magnification images show the Schwann cellsclosely associated with the neurites to the extent that they align alongwith and adjacent to the βIII-tubulin positive extensions (FIGS. 23d-f). These results were well correlated to observations of the DRGcells inside the hydrogel as observed by phase contrast microscopy(FIGS. 22 a-b).

Nerve Growth Factor Treatments: Experiments to examine the influence ofNGF in the culture medium versus encapsulated in the hydrogel during itsformation were performed with DRG outgrowth constructs. Three treatmentconditions were compared: a treatment using no NGF (NO-NGF), a treatmentusing free-soluble NGF in the culture medium (FS-NGF), and a treatmentwith enmeshed NGF in the hydrogel network (EN-NGF). Two independentexperiments in each treatment condition were preformed for a total ofsix repeats using two different batches of PEGylated fibrinogenprecursors. The constructs were cultured for four days and imaged dailyto measure the progress of 3-D cell outgrowth from the DRGs into thehydrogel network. Based on results from the phase contrast micrographs(data not shown), the free-soluble and enmeshed NGF (FS-NGF and EN-NGF)facilitated outgrowth of both non-neuronal cells and neurites into thehydrogel as compared to NGF-deprived constructs (NO-NGF). In the absenceof NGF, there was no observable outgrowth of neurites and only partialoutgrowth of non-neuronal cells, which were most likely Schwann cells orfibroblasts. Immunohistochemistry confirms the observations of phasecontrast microscopy in that βIII-tubulin and s100 positive cells werepresent in NGF treatments (FS-NGF and EN-NGF) but only s100 positivecells were seen in the NGF-deprived treatment (NO-NGF) (FIGS. 24 a-c).Based on these qualitative data, it is difficult to conclude if thereare significant differences in 3-D DRG outgrowth between the freesoluble and enmeshed NGF; both free soluble NGF (FS-NGF) and enmeshedNGF (EN-NGF) treatments showed a similar labeling pattern.

In order to further differentiate between the free soluble and enmeshedNGF treatments, quantitative outgrowth experiments were performed. Usingdigital image processing, the distance of neurite outgrowth was measuredin DRG constructs that were cultured with free soluble NGF (FS-NGF) orenmeshed NGF (EN-NGF). FIG. 28 d shows that there was little differencebetween the two treatment conditions at any time during the cultureperiod (p>0.35, n=6). In both free soluble and enmeshed NGF, there was arapid increase in neurite outgrowth over the course of the four-dayexperiment (P<0.01, n=6), with a mean neurite length reaching 719.9 μmand 701.2 μm for FS-NGF and EN-NGF treatments, respectively after fourdays.

Cellular Outgrowth and Hydrogel Biodegradation: Alterations to thebiodegradation properties of the fibrinogen backbone of the hydrogelmatrix can also influence the DRG cellular outgrowth characteristics,particularly as related to the relative invasion of Schwann cells andneurites. Experiments were performed to assess the ability to regulatethe outgrowth kinetics using different compositions of the matrix(relative amount of PEG and fibrinogen) based on the rationale that theproteolytic resistance of the fibrinogen matrix will increase withincreasing concentrations of PEG. Consequently, the PEGylated fibrinogenhydrogels also become more cross-linked with additional PEG, therebychanging the mesh size, hydration and mechanical properties of thematrix. Four different compositions of PEG to fibrinogen were tested,including: 30:1, 60:1, 120:1, and 180:1 (PEG:fibrinogen). It isimportant to note that the composition of the constructs in eachtreatment level was such that the pure PEGylated fibrinogen solution(30:1 treatment) was modified with additional unreacted PEG-DA beforethe UV polymerization step. Two independent experiments in eachtreatment level were preformed for a total of nine repeats using twodifferent batches of pure PEGylated fibrinogen precursors.

Overall, the extent of cellular outgrowth from the DRG into the matrixwas decreased with addition of higher concentrations of PEG-DA in thehydrogel matrix (FIGS. 25 a-p). The lag between neurites and glial cellswas visibly reduced with the addition of higher concentrations of PEG-DA(FIGS. 25 q-t). A summary of the neurite outgrowth kinetics data withthe different concentrations of PEG is summarized in FIG. 25 u.Statistical analysis of the kinetics data (2-factor ANOVA) revealed thatoutgrowth steadily increased with culture time (p<0.01, n=9) and thathigher concentrations of PEG slowed down the cellular invasion (p<0.01,n=9). In particular, constructs made with high concentrations of PEG-DA(120:1 and 180:1) delayed neurite outgrowth significantly from day 2 ofculture when compared to constructs made with lower concentrations ofPEG (30:1 and 60:1). Neurite outgrowth in the 180:1 hydrogels exhibitedslowest outgrowth kinetics of all treatment levels, and reached a meanneurite length of 201.6 μm following four days of culture. Constructsmade with 120:1 exhibited moderate neurite outgrowth rate and reached amean neurite length of 490.8 μm following four days. There was nosignificant difference in neurite outgrowth between the 30:1 and 60:1treatment levels (p>0.50, n=9); in both cases, outgrowth progressed mostrapidly and reached a mean neurite length of 807.8 μm and 850.6 μm,respectively following four days of culture.

Fibrinogen and DRG Cellular Outgrowth: The importance of the fibrinogenbackbone in enabling cellular outgrowth from the DRG into the PEGylatedfibrinogen matrix was investigated using PEG-only hydrogels as controls.DRG constructs were made of 10% PEG-DA without fibrinogen and comparedto constructs made with PEGylated fibrinogen. The constructs werecultured for three days and cellular outgrowth was documented on thethird day of culture. FIG. 26 a shows that without fibrinogen, very fewneurites extend out of the DRG and outgrowth of non-neuronal cells,including Schwann cells, was not observed. In contrast, fibrinogencontaining hydrogels exhibit massive DRG outgrowth, including neuriteand Schwann cell invasion, following three days of culture (FIG. 26 b).These results demonstrate fibrinogen's role in permitting DRG outgrowththat includes proteolytic susceptibility, inductive and conductiveenvironmental cues which may be crucial for functional peripheral nerveregeneration. Consequently, neuronal outgrowth was practicallyeliminated even in the PEGylated fibrinogen hydrogels when DRG cultureswere deprived of NGF (NO-NGF), whereas other cell types (includingSchwann cells) are observed invading the hydrogel (FIG. 26 c).

Analysis and Discussion

Peripheral nerve regeneration is a complex, highly regulated processwhich requires specific environmental cues that are provided by theextracellular matrix (ECM) and the tight bi-directional communicationbetween regenerating axons and their associated Schwann cells. Manyperipheral nerve regeneration strategies using NGCs have been designedto provide the optimal milieu for PNS regeneration, using natural orsynthetic materials and different growth factor delivery strategies.Because NGCs have yet to achieve the efficacy of the nerve autografts,alternative approaches are sought that can leverage the natural healingmechanisms of peripheral nerve repair following moderate injury. To thisend, the PEGylated protein hydrogels of the present invention can serveas a template for nerve regeneration which combines the paracrineeffects of fibrin(ogen) and the control over biodegradation andbioactivity afforded by the PEGylation paradigm.

The experiments descibed hereinabove support a potential NGC biomaterialsystem based on PEGylated fibrinogen hydrogels that maintain outgrowthof DRG cells. A 3-D hydrogel matrix composed of PEG and fibrinogen wasused to encapsulate chicken embryo DRGs to form transparent constructsthat enable straightforward monitoring of the DRG outgrowth (FIGS. 21a-c). Outgrowth of neurites and non-neuronal (glial) cells was observedfrom the DRG into the hydrogel (FIGS. 22 a-d). Furthermore, in vivo likespatial organization of these cells was observed. Specifically, the longneurites were observed in close proximity to their associated glialcells. Using antibodies specific for βIII-tubulin and s100, it was shownthat the s100-positive Schwann cells are highly associated with theradially extending βIII-tubulin positive neurites (FIG. 23 a-f). Theseneuron-Schwann cell complexes enable the production and organization ofmyelin along the length of extending axons in order to provide rapid andefficient propagation of action potentials along axons. Consequently,this distinct spatial organization is a prerequisite for axonalmyelination during the later phase of neuronal regeneration.

It is likely that DRG neurites and glial cells employ a proteolyticmechanism to invade the PEGylated fibrinogen hydrogel matrix in as muchas the hydrogel is highly susceptible to proteases [Almany L, et al.Biomaterials 2005;26(15):2467-77] and is otherwise too dense to permitcellular invasion in the absence of proteolysis. Because the fibrinogenbackbone affords the biosynthetic hydrogel its biodegradability, it alsoprovides a means of releasing cleaved fragments of fibrinogen from thematrix upon degradation. In this manner the kinetics of neurite andglial cell invasion as well as the bioactivity of the releasedfibrinogen fragments can be controlled by changing the relative amountof PEG and fibrinogen. Higher amounts of PEG reduce the susceptibilityto proteolytic degradation of the fibrinogen backbone [Dikovsky D, etal, Biomaterials 2006;27(8):1496-506] and presumably reduces the overallbioactivity of the degraded fibrinogen fragments that are released tothe surrounding tissue [Hooftman G, et al. J Bioact Compat Polym 1996;11:135-159]. Indeed, the addition of PEG to the biosynthetic hydrogelslows down the invasion of both Schwann cells and neurites from the DRG(FIGS. 25 a-p). Furthermore, it appeared that in lower concentrations ofPEG, the non-neuronal outgrowth from DRGs lagged behind the neuriteextensions, whereas the higher concentrations of PEG minimized this lag(FIGS. 25 q-t).

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims. All publications, patents and patentapplications and GenBank Accession numbers mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent or patent application or GenBank Accession numberwas specifically and individually indicated to be incorporated herein byreference. In addition, citation or identification of any reference inthis application shall not be construed as an admission that suchreference is available as prior art to the present invention.

1. A composition-of-matter comprising a synthetic polymer attached todenatured fibrinogen or a therapeutic portion of said denaturedfibrinogen.
 2. The composition of matter of claim 1, wherein saidtherapeutic portion of said denatured fibrinogen is an enzyme cleavageproduct of said denatured fibrinogen.
 3. The composition-of-matter ofclaim 2, wherein said enzyme is selected from the group consisting ofplasmin, collagenase and trypsin.
 4. The composition-of-matter of claim1, wherein said therapeutic portion of said denatured fibrinogen issynthetic.
 5. The composition-of-matter of claim 1, wherein saidtherapeutic portion of said denatured fibrinogen is as set forth in SEQID NO: 1, 2, 3, 4, 5, 6, 7 or
 8. 6. The composition-of-matter of claim1, wherein said synthetic polymer is selected from the group consistingof polyethylene glycol (PEG), Hydroxyapatite/polycaprolactone (HA/PLC),polyglycolic acid (PGA), Poly-L-lactic acid (PLLA), Polymethylmethacrylate (PMMA), polyhydroxyalkanoate (PHA), poly-4-hydroxybutyrate(P4HB), polypropylene fumarate (PPF), polyethylene glycol-dimethacrylate(PEG-DMA), beta-tricalcium phosphate (beta-TCP) and nonbiodegradablepolytetrafluoroethylene (PTFE).
 7. The composition-of-matter of claim 6,wherein said PEG is selected from the group consisting of PEG-acrylate(PEG-Ac) and PEG-vinylsulfone (PEG-VS).
 8. The composition-of-matter ofclaim 7, wherein said PEG-Ac is selected from the group consisting ofPEG-DA, 4-arm star PEG multi-Acrylate and 8-arm star PEG multi-Acrylate.9. The composition-of-matter of claim 8, wherein said PEG-DA is a 4-kDaPEG-DA, 6-kDa PEG-DA, 10-kDa PEG-DA, 14-kDa PEG-DA and/or 20-kDa PEG-DA.10. The composition-of-matter of claim 8, wherein a molar ratio betweensaid PEG-DA to said denatured fibrinogen or said therapeutic portion is2-400 to
 1. 11. The composition-of-matter of claim 8, wherein a molarratio between said PEG-DA to said fibrinogen or said therapeutic portionis 25 to
 1. 12. The composition-of-matter of claim 8, wherein a molarratio between said PEG-DA to said fibrinogen or said therapeutic portionis 75 to
 1. 13. The composition-of-matter of claim 8, wherein a molarratio between said PEG-DA to said fibrinogen or said therapeutic portionis 150 to
 1. 14. The composition-of-matter of claim 1, furthercomprising a pharmaceutical agent.
 15. A scaffold comprising thecomposition-of-matter of claim
 1. 16. A hydrogel comprising acomposition-of-matter which comprises a synthetic polymer attached todenatured fibrinogen or a therapeutic portion of said denaturedfibrinogen.
 17. The hydrogel of claim 16, wherein said naturallyoccurring protein is whole denatured fibrinogen and whereas aconcentration of said units in said hydrogel is selected from a range of0.5-35%.
 18. The hydrogel of claim 16, wherein said denatured fibrinogenis fragmented denatured fibrinogen and whereas a concentration of saidunits in said hydrogel is selected from a range of 0.5-35%.
 19. Thehydrogel of claim 18, wherein modulus of elasticity of said hydrogel isin a range of 0.02-0.11 kPa for 10-20% polymer.
 20. The hydrogel ofclaim 19, wherein modulus of elasticity of said hydrogel is in a rangeof 0.01-0.07 kPa for 10-20% polymer.
 21. A medical device comprising acomposition-of-matter which comprises a synthetic polymer attached todenatured fibrinogen or a therapeutic portion of said denaturedfibrinogen.
 22. The medical device of claim 21 is an intracorporealdevice.
 23. The medical device of claim 21 is an extracorporeal device.24. The medical device of claim 21 is selected from the group consistingof a prosthetic device, a pacemaker, an artificial joint, a heart valvereplacement, temporary implant, a permanent implant, a stent, a vasculargraft, an anastomotic device, a clamp, an aneurysm repair device and anembolic device.
 25. A pharmaceutical composition comprising acomposition-of-matter which comprises a synthetic polymer attached todenatured fibrinogen or a therapeutic portion of said denaturedfibrinogen.
 26. The pharmaceutical composition of claim 25, wherein saidtherapeutic portion of said denatured fibrinogen is an enzyme cleavageproduct of said denatured fibrinogen.
 27. The pharmaceutical compositionof claim 26, wherein said enzyme is selected from the group consistingof plasmin, collagenase and trypsin.
 28. The pharmaceutical compositionof claim 25, wherein said therapeutic portion of said denaturedfibrinogen is synthetic.
 29. The pharmaceutical composition of claim 25,wherein said therapeutic portion of said denatured fibrinogen is as setforth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7 or
 8. 30. The pharmaceuticalcomposition of claim 25, wherein said synthetic polymer is selected fromthe group consisting of polyethylene glycol (PEG),Hydroxyapatite/polycaprolactone (HA/PLC), polyglycolic acid (PGA),Poly-L-lactic acid (PLLA), Polymethyl methacrylate (PMMA),polyhydroxyalkanoate (PHA), poly-4-hydroxybutyrate (P4HB), polypropylenefumarate (PPF), polyethylene glycol-dimethacrylate (PEG-DMA),beta-tricalcium phosphate (beta-TCP) and nonbiodegradablepolytetrafluoroethylene (PTFE).
 31. The pharmaceutical composition ofclaim 30, wherein said PEG is selected from the group consisting ofPEG-acrylate (PEG-Ac) and PEG-vinylsulfone (PEG-VS).
 32. Thepharmaceutical composition of claim 31, wherein said PEG-Ac is selectedfrom the group consisting of PEG-DA, 4-arm star PEG multi-Acrylate and8-arm star PEG multi-Acrylate.
 33. The pharmaceutical composition ofclaim 32, wherein said PEG-DA is a 4-kDa PEG-DA, 6-kDa PEG-DA, 10-kDaPEG-DA and/or 20-kDa PEG-DA.
 34. The pharmaceutical composition of claim32, wherein a molar ratio between said PEG-DA to said denaturedfibrinogen or said therapeutic portion is 2-400 to
 1. 35. Thepharmaceutical composition of claim 32, wherein a molar ratio betweensaid PEG-DA to said fibrinogen or said therapeutic portion is 25 to 1.36. The pharmaceutical composition of claim 32, wherein a molar ratiobetween said PEG-DA to said fibrinogen or said therapeutic portion is 75to
 1. 37. The pharmaceutical composition of claim 32, wherein a molarratio between said PEG-DA to said fibrinogen or said therapeutic portionis 150 to
 1. 38. The pharmaceutical composition of claim 25, furthercomprising a pharmaceutical agent.
 39. A method of treating a disordercharacterized by a tissue damage, the method comprising providing to asubject in need-thereof a composition which comprises a syntheticpolymer attached to denatured fibrinogen or a therapeutic portion ofsaid fibrinogen, said composition being formulated for releasing saidtherapeutic portion of said fibrinogen in a pharmacokineticallyregulated manner, thereby treating the disorder characterized by tissuedamage or malformation.
 40. The method of claim 39, wherein saidpharmacokinetically regulated manner is immediate releasing of saidtherapeutic portion of said fibrinogen.
 41. The method of claim 40,wherein said pharmacokinetically regulated manner is sustained releasingof said therapeutic portion of said fibrinogen.
 42. The method of claim41, wherein said sustained releasing of said therapeutic portion of saidfibrinogen is between 1 week and 200 weeks.
 43. The method of claim 42,wherein said composition is comprised in a scaffold, medical device,pharmaceutical composition or a hydrogel.
 44. The method of claim 39,wherein said composition is formulated for local administration.
 45. Themethod of claim 39, wherein said composition is formulated for systemicadministration.
 46. The method of claim 39, wherein said therapeuticportion of said denatured fibrinogen is an enzyme cleavage product ofsaid denatured fibrinogen.
 47. The method of claim 46, wherein saidenzyme is selected from the group consisting of plasmin, collagenase andtrypsin.
 48. The method of claim 39, wherein said therapeutic portion ofsaid denatured fibrinogen is synthetic.
 49. The method of claim 39,wherein said therapeutic portion of said denatured fibrinogen is as setforth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7 or
 8. 50. The method of claim39, wherein said synthetic polymer is selected from the group consistingof polyethylene glycol (PEG), Hydroxyapatite/polycaprolactone (HA/PLC),polyglycolic acid (PGA), Poly-L-lactic acid (PLLA), Polymethylmethacrylate (PMMA), polyhydroxyalkanoate (PHA), poly-4-hydroxybutyrate(P4HB), polypropylene fumarate (PPF), polyethylene glycol-dimethacrylate(PEG-DMA), beta-tricalcium phosphate (beta-TCP) and nonbiodegradablepolytetrafluoroethylene (PTFE).
 51. The method of claim 50, wherein saidPEG is selected from the group consisting of PEG-acrylate (PEG-Ac) andPEG-vinylsulfone (PEG-VS).
 52. The method of claim 51, wherein saidPEG-Ac is selected from the group consisting of PEG-DA, 4-arm star PEGmulti-Acrylate and 8-arm star PEG multi-Acrylate.
 53. The method ofclaim 52, wherein said PEG-DA is a 4-kDa PEG-DA, 6-kDa PEG-DA, 10-kDaPEG-DA and/or 20-kDa PEG-DA.
 54. The method of claim 52, wherein a molarratio between said PEG-DA to said denatured fibrinogen or saidtherapeutic portion is 2-400 to
 1. 55. The method of claim 52, wherein amolar ratio between said PEG-DA to said fibrinogen or said therapeuticportion is 25 to
 1. 56. The method of claim 52, wherein a molar ratiobetween said PEG-DA to said fibrinogen or said therapeutic portion is 75to
 1. 57. The method of claim 52, wherein a molar ratio between saidPEG-DA to said fibrinogen or said therapeutic portion is 150 to
 1. 58.An article-of-manufacturing comprising a packaging material and thecomposition of claim 1 identified for treating a disorder characterizedby a tissue damage or malformation.